Harnessing Antibody-Dependent Cellular Cytotoxicity To Control HIV

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Harnessing antibody-dependent cellular cytotoxicity to control HIV-1 infection Nizar Mohammad Abuharfeil, Mahmoud Yaseen, and Fawzi M Alsheyab ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00167 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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ACS Infectious Diseases

Title: Harnessing antibody-dependent cellular cytotoxicity to control HIV-1 infection Running title: ADCC and HIV-1 control Nizar Mohammad Abuharfeil1*, Mahmoud Mohammad Yaseen2*#, Fawzi M. Alsheyab1

1Department

of Applied Biological Sciences, College of Science and Arts, Jordan

University of Science and Technology, Irbid, 22110, Jordan 2Department

of Medical Laboratory Sciences, Faculty of Applied Medical Sciences,

Jordan University of Science and Technology, Irbid, 22110. Jordan

*Equal

contribution.

#Corresponding

Mahmoud

author:

Mohammad

Yaseen,

E-mails:

[email protected]

or

[email protected] Phone (Mobile): 00962-786537563

1 ACS Paragon Plus Environment

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Passive administration of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (bNAbs) has been recently suggested as a promising alternative therapeutic approach for HIV-1 infection. Although the success behind the studies that used this approach has been attributed to the potency and neutralization breadth of anti-HIV-1 antibodies, several lines of evidence support the idea that specific antibody-dependent effector functions, particularly antibody-dependent cellular cytotoxicity (ADCC), play a critical role in controlling HIV-1 infection. In this review, we showed that there is a direct association between the activation of ADCC and better clinical outcomes. This, in turn, suggests that ADCC could be harnessed to control HIV-1 infection. To this end, we addressed the passive administration of bNAbs capable of selectively activating ADCC responses to HIV-1 patients. Finally, we summarized the potential barriers that may impede the optimal activation of ADCC during HIV-1 infection and provided strategic solutions to overcome these barriers. KEY WORDS: Natural killer (NK) cells; Passive antibody administration; Fc-FcγR; Hypo-/defucosylated Fc broadly neutralizing anti-HIV-1 antibodies (bNAbs), HIV-1 controllers; Env conformation.


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Finding a cure for HIV-1 infection is still a global major goal of research on HIV-1. Such a cure can be sterile or functional according to the viral eradication or suppression scenarios, respectively. A sterile cure can only be achieved when the virus is completely eradicated from HIV-1 infected patients, which is, however, unlikely to be achieved by the currently available antiretroviral therapy (ART) because it cannot eradicate HIV-1 latent reservoirs. On the other hand, a functional cure attempts to maintain HIV-1 replication suppressed in undetectable level for prolonged periods of time after cessation of short-term therapy, also called sustained virologic remission or post-treatment control1. In fact, maintaining HIV-1 suppressed in undetectable level is possible by available ART, but achieving sustained virologic remission after cessation of ART is very difficult even if HIV-1 viremia has been suppressed for prolonged periods of time2-5. HIV-1 persists because of the stable latent reservoir which is invisible to the immune system and currently available ART, further, the currently ART puts pressure directly on HIV-1 replication instead of targeting immune system to enhance its responses against HIV-1 and, thus, cessation of therapy leads to gradual emergence of viruses from the reservoir. This, in turn, necessitates continuing research to find new alternative and feasible therapeutic approaches target the virus while enhancing immune responses6. Indeed, antibody-therapy may provide one such alternative therapeutic approach for HIV-1 infection6. As a bi-functional weapon, an antibody can mediate its therapeutic or preventive effects by one of two mechanisms. First, it can bind to and neutralize target(s) via its variable antigen-binding fragment (Fab) regions, and second, it can mediate effector functions via the engagement of its constant crystallizable fragment (Fc) region with the corresponding Fc receptor (FcR) expressed on effector immune cells. In the case of HIV-1 infection, literature shows a remarkable interest in 3 ACS Paragon Plus Environment


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studying the neutralization property over the effector functions mediated by anti-HIV1 antibodies. Hence, several pre-clinical and clinical investigations have been conducted to assess the ability of neutralizing anti-HIV-1 antibodies to protect and/or control HIV-1 infection6-9. In the context of HIV-1 infection control, initial investigations failed to confirm the efficacy of bNAbs (i.e. 2G12, 2F5, b12, and 4E10) to control HIV-1 viremia. However, the recent discovery of extremely potent antibodies with much more neutralization breadth than those initially used in pre-clinical and clinical studies has renewed the attention to evaluate their efficacy in controlling HIV1 viremia (reviewed in 6). Interestingly, for the first time in non-human primates and humans, investigators have demonstrated the efficacy of such antibodies in controlling HIV-1 viremia. Shortly thereafter, sequential successes in both pre-clinical and clinical studies have also been achieved using passive administration strategy of bNAbs10-12. These studies have mainly attributed the success of passively administered bNAbs in controlling HIV-1 viremia to their extraordinary neutralization breadth6. Nonetheless, several lines of evidence, directly or indirectly, have suggested that the achieved HIV1 control using this strategy, at least in part, was attributed to their mediated effector functions upon the engagement of anti-HIV-1 antibodies with the effector innate immune cells through Fc-Fc-gamma receptor (FcγR) interaction13-19. This indicates that antibody-dependent effector functions could be essential for viral control. As such, in this review we sought to address (i) the therapeutic potential of antibody-mediated effector functions, particularly the ADCC activity, in controlling HIV-1 infection; (ii) the factors that influence such immune responses; and (iii) the viral evasion strategies. These will provide valuable insights into the future design of an effective HIV-1 therapeutic strategy (functional cure) that may even lead to a sterile cure.


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Lastly, it is worthy to point out that there are different antibody-dependent effector functions that can be mediated against HIV-1. These include: ADCC, antibodydependent cellular phagocytosis, and antibody-dependent complement cytotoxicity (classical pathway). In this paper, we aim to address: (i) the role of ADCC during the course of HIV-1 infection in a chronological view; (ii) the therapeutic potential of this approach; and (iii) the barriers that may hurdle the success of this potential therapeutic approach for HIV-1 infection.

ADCC

RESPONSES

AND

HIV-1

DISEASE

PROGRESSION:

A

CHRONOLOGICAL VIEW The evidence for the negative correlation between HIV-1 disease progression and ADCC responses arose from the early investigations in this area of HIV-1 research. More than two decades ago, Ahmad and colleagues demonstrated that the higher CD4+ T cells count (a critical predictor of HIV-1 disease progression) is associated with the higher plasma anti-HIV-1 gp120/gp41-specific antibodies capable of activating ADCC responses (ADCC-Abs)20. Baum and colleagues have evaluated the ADCC activity of ADCC-Abs in the serum obtained from different groups of HIV-1 infected individuals, each of which with a distinct clinical status (rapid-, normal-, and non-progressors), at different time points over a decade21. Their results indicated that HIV-1 infected individuals with high titers of ADCC-Abs did not progress to AIDS when compared to individuals with lower antibody titers. Importantly, when they studied the longitudinal changes at the individual-level there was even a stronger correlation between the titers of ADCC-Abs and slower disease progression. Although, the results of this study were in agreement with some previous studies22, 23, they were in disagreement with other 5 ACS Paragon Plus Environment


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studies that failed to find such a relationship24, 25. Indeed, this was attributed to the variation in experimental conditions that include but not limited to: (i) different ADCC assays used; (ii) the nature of target cells used; (iii) the nature and expression level of antigen used on target cells; (iv) the use of different HIV-1 isolates; and (v) the capacity of used immune effector cells to mediate ADCC. Of note, several early studies have reported that the titers of ADCC-Abs are decreased as the disease progresses20, 22, 26, indicating that ADCC-Abs declining is a consequence of, but may also cause, disease progression. As such, it has been shown that using sera-containing anti-HIV-1 antibodies obtained from symptomatic HIV-1 (AIDS) patients to drive ADCC activity by normal NK cells against HIV-1 gp120expressing target cells frequently failed to mediate ADCC by normal NK cells27. This finding indicates that antibodies from AIDS patients are defective or, at least, notcapable of activating ADCC. The later can be attributed to the presence of immune complexes, or relatively high levels of non-ADCC-activating IgG isotypes (i.e. IgG2 and/or IgG4) in AIDS patients' sera, taking into consideration that IgG1 and IgG3 are the major ADCC-Abs isotypes in humans26-28. This, in turn, may reflect the existence of competition between ADCC-mediating and non-ADCC-mediating IgG isotypes in binding to the HIV-1 epitopes expressed on HIV-1 infected cells. Moreover, the competition in binding to the FcγR expressed on effector immune cells due to the presence of immune complexes may modulate the ADCC activity during HIV-1 infection. On the other hand, transfusion of plasma containing anti-HIV-1 antibodies obtained from asymptomatic HIV-1 infected individuals to AIDS patients as a therapeutic strategy has resulted in a partial success29. In other words, this transfusion delayed the disease progression and decreased the viral load in some, but not all, 6 ACS Paragon Plus Environment

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cases29. Nevertheless, a study conducted by Skowron and coworkers in 1997 showed a direct correlation between disease progression and ADCC-Abs activity. Their results indicated that ADCC responses participate in CD4+ T cells depletion during the course of HIV-1 infection, thereby, enhancing the disease progression30. To explain this controversy, we would say that such experimental conditions cannot lead to such conclusive results, at least, due to two reasons: first, the conclusions were totally based on the capability of effector immune cells from HIV-1 infected individuals to mediate ADCC responses against HIV-1 gp120-coated target cells. Indeed, we can logically expect that exposing gp120-coated cells to ADCC-cells in the presence of anti-gp120 ADCC-Abs will lead to the activation of ADCC responses and thereby mediating the killing of target cells regardless their type. Second, this study has not determined the ADCC-Abs titers, and thus, a negative association between ADCC-Abs titers and CD4+ T cells depletion cannot be made. These data reflect the importance of ADCC-Abs in controlling HIV-1 infection. However, the presence of ADCC-Abs alone is not sufficient to mediate ADCC responses, because such immune responses require the presence of specific effector immune cells capable of interacting with antibodies in a manner dependent on Fc-FcγR interaction. Monocytes and NK cells are the major effector immune cells involved in ADCC (ADCC-cells) activation during HIV-1 infection27,

31-33.

Of note, although

monocytes/macrophages and to a less extent neutrophils can mediate ADCC responses34, the subsequent discussions will focus on NK cells because they are the principal mediators of ADCC responses and most investigations in this area of research mainly focused on NK cells. However, this does not mean to exclude the potential role of monocytes/macrophages and neutrophils in ADCC mediation, for more details see Ref.34,

35.

Importantly, several studies have demonstrated that the functionality of 7 ACS Paragon Plus Environment


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ADCC-cells from HIV-1 infected individuals is significantly reduced as compared to the effector cells from uninfected individuals20, 27, 32, 36. Noteworthy, this functional defect was partially reversed by the in vitro treatment with physiological concentrations of recombinant Interluekin-2 (IL-2) and Interferon-gamma (IFN- γ)20. As such, adoptive transfer of in vitro activated and expanded NK cells into AIDS patients was shown to be beneficial37-39. This suggests that transfusion of competent-ADCC-cells into HIV-1 infected individuals with defects in their ADCC-cells could benefit them in this context. At that time, most studies have succeeded to establish the evidence for the critical correlation between ADCC responses and controlling HIV-1 infection. This explains why Ahmad and Menezes, more than two decades ago, suggested using passive administration of ADCC-Abs along with competent-ADCC-cells as a therapeutic strategy for HIV-1 infection38. Later studies have also confirmed and extended these notions. For example, in 1999, Forthal and his coworkers observed a critical correlation between ADCC responses and survival in severely compromised AIDS patients40. They also indicated that ADCC responses and perhaps NK cells function act as predictors of survival in late disease stages, nonetheless, they did not exclude their role in less compromised patient states40. In 2001, other studies observed a direct correlation between the serum titers of ADCC-Abs and the count of circulating CD4+ T cells and indirect correlation with the plasma viral load in HIV-1 infected individuals, supporting the positive impact of ADCC responses in controlling HIV-1 infection41, 42. Of note, Forthal and coworkers were able to find a direct correlation between the titers of ADCC-Abs and viral load in patients with high number of CD4+ T cells only42, suggesting a critical role for CD4+ T cells in maintaining ADCC-Abs.


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They also indicated that CD4+ T cells may augment the function of effector immune cells involved in ADCC mediation by secreting cytokines such as IL-2 and IFN-γ. In an attempt to determine the impact of ADCC responses on the partial control of HIV-1 viremia observed during the acute phase of infection, a study was conducted in 200143. The results of this study strongly supported the role of ADCC responses in the partial viremia control during the acute phase of HIV-1 infection. This is because ADCC-Abs that target HIV-1 infected cells were detectable in the majority of HIV-1 infected individuals during the early days of infection, including the period of viremia declining. Moreover, the magnitude of ADCC responses was shown to be inversely correlated with the plasma viral load; hence, investigators suggested that using HIV-1 antigens that elicit ADCC-Abs may offer an avenue to design an effective HIV-1 vaccine43. These results were later supported by several studies44, 45. To further support the role of ADCC responses in controlling HIV-1 infection, the results obtained from longitudinal in vivo studies on simian immunodeficiency virus (SIV)-infected macaques, a non-human primate model of HIV-1 infection, have shown that the ADCC activity is directly associated with the delayed disease progression46. Other studies compared the impact of different antibody responses on the clinical status of HIV-1 infection based on their prevalence in the sera from HIV-1 patients47. One major finding of these studies was that HIV-1 infection induces various types of antibodies exhibiting different effector functions including: neutralizing, ADCC-mediating, and infectionenhancing anti-HIV-1 antibodies. Both the neutralizing and ADCC-Abs titers were lower in the sera obtained from HIV-1 infected individuals with low CD4+ T cells number. Remarkably, infection-enhancing anti-HIV-1 antibodies, which are, however, unwanted consequence of HIV-1 infection, were shown to be dependent on the existence of complement proteins. The presence of complement proteins was also 9 ACS Paragon Plus Environment


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shown to reduce the capacity of neutralizing anti-HIV-1 antibodies to neutralize viral isolates from sera positive for neutralizing anti-HIV-1 antibodies. This may suggest that neutralization assays should not be conducted in the absence of complement proteins to be of physiological relevance, in part, because they are ubiquitously expressed in vivo. At that time the infection-enhancing anti-HIV-1 antibodies were known to be directed against two immunodominant-domains of HIV-1 gp41, while neutralizing antiHIV-1 antibodies were known to be directed against CD4-binding sites and variable domains of the viral glycoproteins48-50, indicating that the effector antibody responses against HIV-1 may, in part, be dependent on the targeted viral epitope(s) (discussed later). As such, Subbramanian and coworkers stated that such notions could provide a guide to design an effective HIV-1 vaccine in future47. In fact, studies in this area of HIV-1 research were not only restricted to investigate ADCC responses in blood circulation, but also were extended to include other compartments such as cervical-vaginal tissues51,

52.

These studies have

demonstrated that ADCC responses in female genital tract inversely correlate with the viral load, underscoring the strong role of ADCC responses in controlling HIV-1 viremia in such compartments, which, in turn, may decrease HIV-1 transmission to partners. In 2003, Chuenchitra and colleague failed to find a significant difference in ADCC activity between two clinically different groups of HIV-1 patients (disease progressors and slow disease progressors) in their longitudinal study53. Indeed, this was attributed to a limitation in the criteria that used to determine the ADCC activity. In other words, authors' findings were completely based on the ability of serum obtained from the two clinically different groups of HIV-1 infected individuals to mediate ADCC responses after being cultured with competent-ADCC-cells obtained from 10 ACS Paragon Plus Environment

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healthy individuals. Such an experimental condition can only be used to determine the differences between HIV-1 progressors and slow-disease progressors in their abilities to induce the production of ADCC-Abs, but not to determine the ADCC responses in total, because such responses are dependent on the presence of both the ADCC-Abs and functional ADCC-cells, as aforementioned. However, several years later, particularly in 2009, Lambotte and coworkers who attempted to determine the spectrum of anti-HIV-1 antibodies in HIV-1 controllers found a significant correlation between the levels of ADCC-Abs, but not other types of antibodies, and viral control in HIV-1 controllers when compared to viremic patients54. Interestingly, although they found similar or lower levels of all antibody types (HIV-1 binding and neutralizing antibodies) in HIV-1 controllers versus HIV-1 viremic individuals, ADCC-Abs were present in a greater magnitude in HIV-1 controllers. Furthermore, to delineate whether the control of HIV-1 infection was not driven by other immune responses such as those mediated by CD8+ T responses, which are known to be associated with the viral control in HIV1 patients, they measured HIV-1-specific CD8+ T cells responses. Notably, they observed that ADCC responses were present in some HIV-1 patients who exhibit low HIV-1-specific CD8+ T cell responses, suggesting that ADCC responses are capable to drive HIV-1 infection control independent of CD8+ T cells responses. In 2013, using intracellular cytokine staining ADCC assay to measure IFN-γ and fluorescent antibodies to detect CD107a (a degranulation marker) on NK cells, Wren et al. showed that broader ADCC responses were associated with slower HIV-1 disease progression in long-term slow-progressors cohort when compared to non-longterm slow-progressors cohort55. Although ADCC activity against HIV-1 is generally assessed against the viral envelop glycoproteins (Env), Wren and colleagues have found a significant number of ADCC responses against non-Env viral proteins in the sera of 11 ACS Paragon Plus Environment


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long-term slow-progressors. Another study by a team of investigators from China have evaluated the correlation between ADCC responses and disease progression in a cohort of untreated chronically HIV-1 infected former plasma donor by examining the NK cells abundance and functions56. Regarding the total number of NK cells, unlike elite controllers who had no significant reduction in the total NK cells count as compared to healthy subjects, there was a significant reduction in non-controller groups. Furthermore, ADCC responses were shown to be negatively associated with the HIV1 disease progression and correlated positively with specific human leukocyte antigen (HLA) alleles, yet the exact mechanism by which specific HLA-alleles exhibiting ADCC responses responsible for the controlling of HIV-1 viremia is not well understood. Lambotte et al. using GranToxiLux (GTL) ADCC assay, have also shown that HIV-1 controllers express significantly higher ADCC responses compared to viremic patients, in addition, they have observed that ADCC-Abs titers were significantly higher in HLA B57-negative controllers compared to HLA-B57-positive controllers57. Indeed, Lambotte and colleagues have found that ADCC activity positively correlates with plasma viral load, suggesting that ADCC responses may be subjected to stimulation by HIV-1 replication. However, the significantly lower ADCCAbs titers in HIV-1 viremic (high viral load) patients has led to another suggestion, that is the development of sufficient ADCC-Abs in HIV-1 controllers requires the presence of additional factor(s) that viremic patients do not have. Perhaps, this requirement is the help of CD4+ T cells, since they play an indispensable role in maintaining efficient HIV-1-specific antibodies producing B cells. Indeed, it is of particular importance to know that the levels and functions of CD4+ T cells and B cells are relatively preserved among HIV-1 controllers58,

59.

Importantly, they have observed that some HIV-1

patients had undetectable titers of ADCC-Abs, these patients are known as elite


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controllers, since they can maintain HIV-1 load in undetectable levels by standard assays for years without treatments, suggesting the presence of additional factor(s) other than ADCC responses that could also participate in HIV-1 infection control. Nonetheless, one explanation for this observation is that the very low amounts of HIV-1 antigens in elite controllers could be insufficient to maintain the production of sufficient ADCC-Abs titers that can be detected by ADCC assays. In an attempt to determine the impact of broad ADCC responses against HIV-1 in controlling HIV-1 infection, Madhavi et al. have conducted their study60. Importantly, they found that HIV-1 controllers exhibit ADCC responses that recognize broad spectrum of HIV-1 Env. Moreover, the magnitude of ADCC responses in HIV-1 controllers was significantly much higher than HIV-1 non-controllers (progressors). While in contrast to the Jia et al.56 findings who indicated that higher ADCC responses were observed in HIV-1 infected patients for greater than one year than those for less than a year, the findings of Madhavi and colleagues indicated that there was no correlation between the duration of infection and ADCC responses60. Moreover, Madhavi et al. findings suggest that ADCC responses to HIV-1 Env commonly target glycan-dependent epitopes60. This, in turn, suggests that glycosylation of HIV-1 Env is critical for ADCC responses. However, one possible limitation of this study in this regard is the differences of glycosylation patterns of HIV-1 Env on infected T cells compared to the used 293-derived cells. From another point of view, Lai and coworkers have investigated the relationship between cellular and humoral adaptive immune responses in controlling HIV-1 infection61. More precisely, they wanted to determine the impact of presence or absence of a protective HIV-1-specific HLA allele on the antibody immune responses. One major finding of this study is that there was no association between divergent antibody responses, including ADCC responses, in HIV13 ACS Paragon Plus Environment


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1 controllers in the presence or absence of protective HLA allele, suggesting that (i) CD8+ T cell responses mediated by HLA alleles do not alter the characteristics of antibody responses in such patients, and (ii) the effector functions of antibodies, including the ADCC activity, can mediate HIV-1 control in the absence of CD8+ T cell responses. Another study has also confirmed the critical impact of ADCC responses on viral control and mortality in infants62. In this study, Milligan et al. have shown that the increased activity of ADCC responses, which results from the passive acquired ADCCAbs, particularly IgG1 antibodies, in HIV-1 infected infants born to HIV-1 infected mothers positively correlates with their survival. To address the association between HIV-1 infected infants' ADCC responses, survival and antibody neutralization activity, they examined the potency and breadth of neutralizing anti-HIV-1 antibodies (NAbs) across HIV-1 infected infants. They have shown that there was a moderate, albeit not significant, association between ADCC activity and breadth and potency of NAbs. There was also a non-significant association between NAbs breadth and infants' survival, supporting the therapeutic potential of passive administration of ADCC-Abs in protecting against HIV-1 disease progression. More recently, Madhavi et al.63 have shown that HIV-1 elite controllers (a rare group of HIV-1 patients who naturally maintains HIV-1 replication suppressed "in undetectable levels" for many years) have higher levels of ADCC-Abs compared to HIV-1 progressors. In line with the previous investigations, a recent study has also confirmed the critical impact of ADCC responses in controlling HIV-1 infection in a cohort of long-term non progressors from India64. Taken together, most studies have indicated that ADCC responses correlate with slow disease progression, suggesting that such immune responses can be implicated for future therapeutic strategies for HIV-1 infection control. 14 ACS Paragon Plus Environment

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PASSIVE BNABS ADMINISTRATION, VIREMIA CONTROL, AND A POTENTIAL ROLE OF ADCC RESPONSES Remarkably, in 2009, two major events were reported regarding anti-HIV-1 antibodies in general. First, the results obtained from a modest (31.2%) but the most successful clinical trial in humans to date, the RV144 clinical trial65. This trial showed a critical correlation between protection against HIV-1 infection and mediation of ADCC responses in vaccine recipients. The second correlate analyses of this trial showed that the low HIV-1 acquisition risk was associated with the induction of high levels of plasma non-neutralizing anti HIV-1 IgG, exhibiting potent ADCC responses, and low IgA antibody titers, underscoring the critical impact of effector functions mediated through Fc-FcR interaction, particularly ADCC responses66-68. This in turn, renewed the attention to evaluate the protective and therapeutic potential of such immune response against acquiring HIV-1 infection. Second, the isolation of new extremely potent and broad neutralizing anti-HIV-1 antibodies. Both events renewed the attention to reevaluate the protective and therapeutic efficacy of such antibodies against HIV-1 infection (reviewed in 6). The efficacy of recently discovered bNAbs in controlling viremia were demonstrated in pre-clinical studies on non-human primates (rhesus macaques) infected with simian-HIV (SHIV)13-15. Of note, Barouch and colleagues have indicated that the functional immune system and intact effector immune cells may underlie the ability of rhesus macaques treated with a single antibody infusion to control viremia when compared to previous studies that used several antibody infusions to treat immunocompromised humanized mice13, 15, suggesting the critical role of antibody mediated effector functions in controlling viremia. In 2014, Bournazos et al.16 revealed that the in vivo activity of bNAbs requires Fc-mediated 15 ACS Paragon Plus Environment


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effector functions. In 2015, Caskey and her colleagues demonstrated the efficacy of bNAbs to control viremia for the first time in humans17. Importantly, these investigators have mentioned an advantage of antibody therapy over the conventional ART, which is the engagement of Fc portion of antibodies with the FcγR expressed on effector immune cells that could enhance the host immunity17. In 2016, the critical role of Fceffector function mediated by bNAbs, particularly ADCC responses, in eliminating HIV-1 infected cells and controlling viremia was also confirmed18, 19. Taken together, in part, these data emphasize that passive administration of bNAbs with potent antibody effector functions, particularly ADCC responses, are directly associated with better clinical outcomes in HIV-1 infection, and therefore could be considered as a potential candidate for a future therapeutic HIV-1 vaccine.

ADCC RESPONSES MEASUREMENT During the past three and half decades, several assays have been developed and used to measure ADCC responses against HIV-1 (recently described in these references69-71. In brief, these assays include: (i) Chromium release ADCC (51CrADCC) assay, in this assay HIV-1 infected or Env-coated target cells are labeled with radioactive

51Cr

and incubated with effector cells and gp120-specific antibodies or

HIV-1 positive sera, ultimately ADCC activity (killing of target cells) is measured by release of radioactive 51Cr into the supernatant of cell culture. (ii) NK cell activation assay, this assay is used to assess the activation of NK cell functions by measuring intracellular IFN-γ, cell surface expression of CD107a or by measuring the loss of intracellular granzyme B, after the incubation of effector cells (NK cells) with the target cells (HIV-1 infected or Env coated-cells) and specific antibodies mixture. (iii) GTL16 ACS Paragon Plus Environment

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ADCC assay, similar to the above mentioned assays that used target cells infected with the virus or coated with the viral Env glycoproteins, however, this assay measures an early step in the target cell lysis pathway mediated by effector cells, which is the delivery of granzyme B to target cells. Target cells are labeled with viability and fluorescent dyes before being incubated with effector cells in the presence of anti-HIV1-specific ADCC-Abs and granzyme B substrate. The induction of ADCC responses means that the effector cells released their granular contents of granzyme B which hydrolyzes the granzyme B substrate upon entrance to target cells resulting in activation of its fluorescence which can be detected by flow cytometry. (iv) Rapid and fluorometric ADCC (RFADCC) assay, in this method target cells are sensitized with intact virus, or inactivated virus or coated with Env glycoproteins, and stained with PKH-26 (a membrane dye) and intracellular carboxyfluorescein diacetate, succinimidyl ester (CSFE) (a cell viability dye). Then, these cells are incubated with HIV-1 specificantibodies and effector cells, and the target cell killing mediated by ADCC activity is measured by emergence of cell population that lost cell viability and positively stained with the membrane dye. (v) Luciferase ADCC assay, the ADCC activity (elimination of target cells) in this method is assessed by measuring the loss of luciferase activity (which is only expressed after productive HIV-1 infection) in HIV-1 infected cells that express luciferase from Tat-inducible promoter after infection. (vi) Infected-cell elimination assay, in this assay the elimination of HIV-1 infected cell mediated by ADCC activity is measured by calculating the percentage loss of HIV-1 infected cells using GFP-expressing viruses or detection of intracellular p24 viral protein. The assessment of ADCC responses can be achieved by measuring the activity of both ADCC-Abs and ADCC-cells. In the case of HIV-1 research, most studies assessed ADCC-Abs activity in HIV-1 infected patients, which is typically measured 17 ACS Paragon Plus Environment


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using chronically infected cell lines, or target cells coated with HIV Env glycoproteins, or peptides. One drawback of these methods is that they measure antibodies that may not direct ADCC against cells infected with primary HIV-1 isolates. The practical consideration is also another drawback of these methods (assays), since such assays rely on NK cells expressing CD16. Nevertheless, the small number of NK cells that can be obtained from a single donor restricts the number of samples that can be processed simultaneously, which requires repeating the isolation of effector cells. The variation in the frequency and NK cells-mediate cytotoxic activity between different donors and the susceptibility of CD4+ T cells HIV-1 infection can also affect the consistency of assays-dependent on primary effector cells. These limitations have encouraged Alpert et al.72 to develop a more practical method to measure the activity of ADCC-Abs. The importance of this method stems from the employing of immortalized NK cell line that express comparable levels of CD16 with the primary cytotoxic NK cells to be of physiological relevant. Noteworthy, this method overcomes the previously mentioned limitations, particularly the practical limitations. On the other hand, it is also of considerable importance to know that sensitizing T cells with inactivated HIV-1 particles, especially those lacking or having defective Nef and/or Vpu viral proteins, or coating them with Env glycoproteins could result in conformational changes in the viral Env as a result of its interaction with the CD4 molecule present on these sensitized T cells (Figure 1)73. This event is to some extent not representative of natural HIV-1 productive infection of target cells, in which Env-CD4 interaction is prevented or at least limited by the viral accessory proteins (Nef and Vpu) (discussed later)73. As a result, this conformational changes in the HIV-1 Env could result in an overestimation the measurement of ADCC responses mediated by certain ADCC-Abs, particularly those targeting the closed Env conformation state such as bNAbs74-77. Additionally,


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certain ADCC-Abs that bind to CD4-binding site (CD4BS) on the viral gp120 compete with the CD4 molecule present on target cells (namely T cells) for binding to CD4BS on gp120, this could also overestimate the measurement of CD4BS antibodies that mediate ADCC activity78, 79. To avoid such unwanted outcomes, the target cells should be carefully selected because they will define the state of Env conformation (type of Env epitopes) that mediate ADCC responses. Other factors may also influence the measurement of ADCC activity such as the binding of soluble gp120, which dissociates from productively infected cells, to CD4 molecule on uninfected cells may sensitize them to ADCC responses mediated by ADCC-Abs

that

recognize

monomeric

CD4-bound

gp120,

and

therefore,

overestimating the total ADCC activity. To avoid such unwanted results, the use of CD4-memitic compounds (CD4mc), which are small molecules that bind HIV-1 gp120 at the Phe 43 cavity near the CD4 binding site and induce conformational changes in the HIV-1 Env similar to those induced by CD4 molecule and sensitize HIV-1 infected cells to ADCC-Abs), can abrogate uninfected cell sensitization by soluble gp12073, since they will compete with CD4 on bystander cells in binding to CD4BS on gp120. Finally, it is also essential to know that recent investigations have shown that ADCC assays that measure ADCC activity mediated through the innate effector cells such as monocytes and NK cells should be carefully selected because certain assays only measure ADCC activity mediated by certain effector cell type. For example, the RFADCC assay, which has been used in several studies in the context of both SIV and HIV-1 infections80-83, primarily has been shown to reflect ADCC responses mediated by monocytes but not NK cells84. Therefore, selection of the ADCC assay should be done carefully.



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HARNESSING ADCC RESPONSES AS AN IMMUNOTHERAPEUTIC APPROACH FOR HIV-1 INFECTION To some extent it is evident that ADCC responses may play a critical role in controlling HIV-1 disease progression during the course of natural infection, and thus could be harnessed as an immunotherapeutic approach to control HIV-1 infection. This stems from different findings which can be summarized as follows: (i) the positive association between the magnitude and breadth of ADCC responses in controlling the disease progression in HIV-1 controllers as compared to HIV-1 progressors (noncontrollers); (ii) the correlate analyses of RV144 trial have shown that the immune response that was responsible for the protection against HIV-1 acquisition in vaccine recipients was mediated via ADCC responses upon induction of non-neutralizing antiHIV-1 antibodies (nNAbs) against V1/V2 domain on HIV-1 gp120; and (iii) the recent preclinical and clinical studies that investigated the efficacy of passively administered bNAbs have suggested that the observed viremia control in these studies was, at least in part, attributed to the antibody-effector functions. These data are of sufficient interest to arise the question of how to mediate the activation of such immune responses in HIV1 patients. Indeed, mediation of ADCC responses depends on the coexistence of three main participants that include: the ADCC-Abs, competent ADCC-cells (mainly NK cells), and target cells (Figure 1). In the context of ADCC-Abs, these antibodies regardless whether are neutralizing or non-neutralizing antibodies should be present in sufficient titers and the magnitude of ADCC-Abs bound to target cells should be equal or above the activation threshold level required to activate ADCC-cells, since the 20 ACS Paragon Plus Environment

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activation/inhibition scenario of ADCC-cells depends on the activating and inhibiting signals. In other words, once the activation signal exceeds the capacity of the inhibitory signal, then the activation process keeps continuing, and vice versa. In the context of antibody effector functions, particularly the ADCC-Abs, these antibodies could be either neutralizing or non-neutralizing, because there is no association between the neutralization activity and ADCC activity of antibodies in general. Essentially, this is because the effector function of a given antibody is influenced by several factors, all of which are related to its Fc region that include: the antibody class (isotype: IgA, IgE, IgG, and IgM; of note although IgM can mediate ADCC responses shortly after infection, the rapid class-switching to IgG class predisposes the IgG class to be the major mediator of ADCC responses in vivo), antibody subclasses (i.e., IgG1, IgG2, IgG3, and IgG4; of note IgG1 and IgG3 are the main subclasses that mediate ADCC responses), and glycosylation of the Fc portion of the antibody. Of note, when talking about neutralization activity of antibodies during the course of natural HIV-1 infection, two types of anti-HIV-1 antibodies could appear (reviewed by us elsewhere6). These antibodies include: (i) the nNAbs, which are the predominant anti-HIV-1 antibodies produced during natural course of infection and appear very early post-HIV-1 infection; (ii) the NAbs that include both the autologousand broadly-neutralizing antibodies. Autologous neutralizing anti-HIV-1 antibodies are developed in most HIV-1 patients several months post-HIV-1 infection, however, the virus rapidly evolves to resist/escape such antibodies. In contrast, bNAbs appear only a minority of HIV-1 patients and their development takes long time (up to years), moreover, the induction of such antibodies by orthodox immunization methods is very difficult, suggesting the superiority of nNAbs over bNAbs in this context.



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Interestingly, there is evidence from both animal- and human-studies that supports a role of nNAbs capable of activating ADCC responses in controlling both the SIV and HIV-1 infections, respectively85, 86. A recent study has suggested that postinfection viremia control could be achieved via passive administration of a mixture of nNAbs with potent Fc-effector functions, including ADCC activity, as seen in nonhuman primates (macaques) challenged with SHIV86. Indeed, the observed viremia control post-infection in this study could be as a result of de novo control of virus replication, which is mediated by Fc effector functions, including ADCC activity. Notably, controlling the viral replication very early post-infection is likely to participate in protecting immune system components and allow the immune responses to be efficiently activated to control the virus replication post-nNAbs decay. This is consistent with the findings from studies on mice, in which that NK cell-mediated killing of target cells early in the immune response triggers a strong CD4+ T cell, CD8+ T cell and humoral immune responses87. These data suggest that nNAbs capable of driving ADCC activity could be harnessed to control HIV-1 infection. However, there is accumulating evidence that HIV-1 can evade ADCC responses mediated by nNAbs through different ways (discussed later). Additionally, recent investigations have revealed that ADCC responses mediated by nNAbs against HIV-1 lack the breadth of recognition of broad spectrum of HIV-1 isolates as bNAbs do88. It has also been revealed that some nNAbs bound to bystander uninfected cells that captured soluble gp120 dissociated from the infected cells and mediate ADCC responses against them. Further, in the context of HIV-1 latently reactivated cells, nNAbs poorly bound to these cells and did not mediated ADCC activity against them88. The nNAbs were also shown to be inefficient to recognize and eliminate infected cells by different transmitted-founder HIV-1 isolates88. 22 ACS Paragon Plus Environment

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In contrast to nNAbs, bNAbs express a high capacity to neutralize a broad spectrum of HIV-1 strains and can efficiently mediate ADCC responses in cell culture88. They also can efficiently target productively infected cells rather than uninfected bystander cells, since they target the closed Env state, underscoring the superiority of bNAbs over nNAbs in mediating ADCC responses in this context88. Additionally, controlling HIV-1 infection by antibodies requires both the neutralization of free HIV-1 particles to prevent new infection and the stimulation of innate effector cells to eliminate already HIV-1 infected cells by means of Fc-FcγR interaction89. As such, harnessing bNAbs with potent capability of activating ADCC responses as a therapeutic approach for HIV-1 infection is suggested. It is of particular interest to know that there is no association between neutralization activity of anti-HIV-1 antibodies and viremia control during natural HIV1 infection. This may be due to the fact that the induction of bNAbs during the course of natural HIV-1 infection takes long time (up to years) and this time is enough for the virus to evolve and escape such bNAbs as has been confirmed by studies that investigated the association between the presence of bNAbs in HIV-1 patients and viremia control (reviewed in 6). An important question that could be asked in this regard that is; if the neutralization activity of bNAbs did not correlate with viremia control from whom these antibodies were isolated, then why the passive administration of bNAbs resulted in viremia control in both animals and human? This mainly could be due to three reasons: first, the extraordinary neutralization potency and breadth of the administered bNAbs. Second, the absence of resistant viral mutants to the administered bNAbs in HIV-1 patients-treated with these antibodies, since the preexistence of resistant mutants was shown to result in the failure of administered antibodies to control viremia6. Further, the emergence of resistant mutants was also shown to limit the 23 ACS Paragon Plus Environment


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efficacy of passive administration of bNAbs as a therapeutic approach. Third, the potent antibody effector functions mediated through the engagement of bNAbs' Fc portion and innate effector cells expressing FcγRs as has been recently revealed by Bournazos et al.16 who showed that the in vivo activity of bNAbs requires Fc-mediated effector functions. This in turn supports the therapeutic potential of bNAbs capable of activating ADCC response. Unfortunately, although the passive administration of bNAbs in both animal models and humans has resulted in viremia control, this control was temporal because of: (i) the declining of antibody titers and/or (ii) the emergence of resistant mutants. The reason behind the emergence of resistant mutants is the high error-prone nature of this virus90 and the use of a single bNAb. To avoid such unwanted outcome, it has been suggested to use combination of bNAbs6. Others have suggested to use bi- or trispecific anti-HIV-1 antibodies or proteins91-95. It is of considerable importance to realize that not all bNAbs are capable to mediate ADCC activity, thus choosing bNAbs capable of mediating ADCC activity is required. Alternatively, enhancing their ADCC activity could be achieved by modification of the Fc region of these antibodies, which in turn could significantly improve the binding to CD16 (FcγRIII) on NK cells. One such a great example of modifications is the glycosylation of the Fc region. Dekkers G et al.96 have recently shown that the Fc-hypo-fucosylation can enhance the binding of IgG1 to CD16 by approximately 17-fold resulting in enhanced natural killer cell-mediated ADCC. They also have shown that Fc-hyper-galactosylation and sialylation (independent of fucosylation) of IgG1 can significantly increase the binding to C1q and thereby enhancing the antibody-dependent complement cytotoxicity. Liu and colleagues97 have also demonstrated that afucosylation of Fc region increases the affinity of Fc to CD16 24 ACS Paragon Plus Environment

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leading to much higher ADCC activity. Furthermore, Forthal et al.98 have shown that the glycosylation of the Fc region of 2G12, a bNAb, significantly influences its binding to FcγR and ADCC activity. Particularly, they have demonstrated that 2G12 glycoforms lacking core fucose can mediate higher ADCC activity against both HIV-1 and SIV infections. Ackerman et al. have also observed that variation in Fc glycosylation critically impacts antiviral activity of anti-HIV antibodies during natural HIV infection99. They have demonstrated that the improved antiviral activity and spontaneous control of HIV infection are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms99. These data indicate that the glycosylation (fucosylation and galactosylation) of the Fc region greatly influences the effector function mediated by antibodies. This, in turn, could be used to optimize the next generation therapeutic antibody applications.

THE OPTIMAL TIME TO ACTIVATE ADCC RESPONSES TO CONTROL HIV-1 INFECTION Generally speaking, during the course of HIV-1 infection immune responses (both the humoral and the cellular immune arms) are gradually attenuated as the disease progresses resulting in various functional defects and alterations in the immune system components that lead to AIDS development in the absence of HIV-1 treatments27, 100, 101.

Among these attenuated immune responses is the ADCC activity. In other words,

the functionality of ADCC responses during the acute phase of HIV-1 is less attenuated as compared to the chronic and AIDS phases27, 100. Indeed, impaired ADCC responses could be as a result of alteration and/or defect(s) in one or both of the ADCC-mediating arms i.e., ADCC-Abs and ADCC-cells, as aforementioned. Therefore, mediating the 25 ACS Paragon Plus Environment


Page 26 of 78

activation of ADCC responses as early as possible post HIV-1 infection is suggested. Importantly, studies have shown that ADCC responses during the acute phase of HIV1 infection are associated with the partial control of viremia43. Moreover, the increased magnitude and broad of ADCC responses during the acute phase of HIV-1 infection have also been shown to be associated with better clinical outcomes44,

45, 102.

Consistently, studies on non-human primates challenged with SHIV have indicated that activation of ADCC responses very early post-infection lead to viremia control86. In addition, passive administration of bNAbs to non-human primates very early can induce long lasting immunity against SHIV infection103. Collectively, these data indicate that the activation of such immune responses during the acute phase of HIV-1 infection is the optimal time point to achieve long-term control of HIV-1 infection. Especially, because the lower viremia during the early phase post-infection is associated with the lower set point viral load, which is a predictor marker of slower disease progression as demonstrated in both animal models and humans104-106. However, this does not mean to exclude the therapeutic potential of such immune responses if activated/mediated during the chronic or even AIDS phases.

POTENTIAL BARRIERS THAT MAY IMPEDE THE ACTIVATION OF ADCC

RESPONSES

DURING

NATURAL

HIV-1

INFECTION

AND

STRATEGIC SOLUTIONS 

Impairment

Of

Effector

Cells

(NK

cells

the

predominant

effectors/mediators of ADCC activity) NK cells are crucial effector innate immune cells that involved in the first line of defense against invaders and abnormal cells (e.g. tumor and viral infected 26 ACS Paragon Plus Environment

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cells). NK cells are heterogeneous cell population that are subdivided into two main subsets based on the expression of certain CD markers, particularly CD56 and CD16 as reviewed by us elsewhere101. The first subset is called CD56bright/CD16dim NK cells and the other one is called CD56dim/CD16bright NK cells. CD56bright/CD16dim NK cells are predominant in the secondary lymphoid tissues accounting for about 90% of NK cells, which are characterized by their ability to secret large amounts of cytokines with limited cytotoxicity, whereas, CD56dim/CD16bright NK cells are highly cytotoxic and predominant in the systemic circulation. Cytotoxic NK cells can mediate their cytotoxic effects on abnormal cells via: (i) activating apoptotic pathways through interaction with FasL and TRAIL; (ii) secreting cytokines that inhibit viral replication; (iii) activating the degranulation process and releasing the contents of their granules (perforin and granzymes) via the engagement of receptors (adhesion receptors, activating receptors, and co-activating receptors) on NK cells with their ligands on target cells, or via the engagement of CD16 (FcγRIIIa) with the Fc portion of antibodies that coat target cells (reviewed in

101).

The later represents the

major killing mechanism mediated by NK cells. However, HIV-1 has many strategies to evade such host defense mechanism. For example, HIV-1 can indirectly down-regulate the expression of CD16 by increasing the expression of certain enzymes such as matrix metalloproteinases (MMPs) on NK cells100 and expand a subset of NK cells population that lacks CD16 (Figure 2)107. HIV1 can also evade ADCC responses via down-regulating the expression of activation receptors such as NKG2A, and NKp46 on NK cells and/or upregulating the expression of inhibitory receptors such as KIR on NK cells, both of which can limit the activation of NK cells to mediate their effector functions,



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including ADCC101. It has also been recently revealed that HIV-1 downregulates the expression of activating receptors ligands such as NKG2D ligand via its Nef protein108. On the other hand, HIV-1 infection can mediate the expansion of anergic subset of NK cells that consequently negatively impacts the cytotoxic activity of other natural killer cell populations109. Additionally, HIV-1 could inhibit the early events of signal transduction pathway upon activation of NK cells through CD16110. Finally, it is worthy to note that the magnitude of NK cells functional defects is directly associated with the disease progression. Therefore, mediating ADDC responses during the early phase of HIV-1 infection is the optimal time to circumvent the viral evasion mechanisms from such immune responses (as discussed earlier). Alternatively, restoring and/or enhancing the functions of NK cells has been suggested by several studies100, 111-113. For instance, Liu et al. have shown that the impaired ADCC activity as a result of decreased CD16 (FcγRIIIa) expression on NK cells in response to an elevation in the levels of MMPs can be restored by inhibiting MMPs100. Consistently, studies on chronically SIV/SHIV infected non-human primates have demonstrated that NK cell-mediated ADCC activity is significantly compromised and this was correlated with the loss of CD16 expression on NK cells112. Importantly, MMPs inhibition was suggested to restore NK cell-mediated ADCC activity. In line with these data, Peruzzi et al. have also demonstrated that the downregulation of CD16 expression on NK cells is associated with ADCC dysfunction, and this immune response can be improved by targeting membrane-type 6 MMP (MT6, also known as MMP25) for inhibition113. These data suggest that using MPPs inhibitors may


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be implicated to restore impaired NK cell-mediated ADDC activity during the course of HIV-1 infection. On the other hand, some studies have shown that cytokines play a critical role in driving NK cell-mediated ADCC activity against HIV-1114. For instance, IL15 and IL-10 were shown to enhanced the ability of NK cells to respond to HIVspecific ADCC-Abs. Other studies have shown that treatment of NK cells obtained from HIV-1 patients with certain cytokines such as IL-21 can enhance their functions, including the ADCC activity, and survival without stimulating HIV-1 replication111. Therefore, manipulating the immunological environment to enhance/restore the potency of NK cell-mediated ADCC activity against HIV-1 could be a promising immunotherapy strategy.



Complement Classical Pathway Activation And Its Impact On ADCC Responses Activation of complement classical pathway is initiated after binding the C1complex (C1q, C1r, and C1s) to the Fc portion of antibody-antigen complex on the targeted membrane115-117. Antibodies differ in their capacities to mediate classical pathway activation, with IgM being best antibody isotype to activate the classical complement pathway. However, since the class-switching to IgG isotype (IgG1, IgG2, IgG3, and IgG4 subclasses) occurs shortly thereafter, these antibodies, but not IgG4, mainly the IgG1 and IgG3 subclasses are responsible to activate the complement classical pathway118,119. The induction of IgG1 and IgG3, but not IgG2 and IgG4, subclasses has been shown to be directly associated with slower HIV-1 disease progression38. These antibodies, IgG1 and IgG3, can also mediate ADCC through interaction of antibody Fc portion with



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the FcγR expressed on different innate immune cells, particularly the NK cells, as aforementioned118, 119. In theory, this indicates that the competition between complement classical pathway and ADCC exists. A study conducted by Mishima et al.119 has shown that the classical pathway activation greatly suppresses the activation of ADCC responses by means of competition. Further, Wang and colleagues have questioned whether the activation of complement system would affect the activation of ADCC or not? Interestingly, their results have shown that complement system activation can disrupt the antibody Fc and FcγR interaction, and also reduce the antibody-induced NK cells activation120, 121. Moreover, the interaction of dendritic cells with complement-opsonized HIV-1 particles was shown to impair their capacity to activate and recruit NK cells by lowering the expression levels of certain chemokines involved in the recruitment of NK cells122. Based on these data we can say that activation of complement classical pathway could limit the activation of ADCC responses against HIV-1 infected cells in vivo by (i) lowering the recruitment and activation of NK cells against HIV-1 infected cells, and (ii) decreasing the availability of Fc regions of antibodies that bind to viral proteins on HIV-1 infected cells. As such, in theory, we can say that the inhibition of complement system, particularly the classical pathway, may improve the activation of ADCC responses (Figure 3). Especially, because the optimal activation of ADCC in the case of HIV-1 was shown to be dependent on the magnitude of ADCC-Abs bound to the viral glycoproteins expressed on the surface of infected cells123. Although theoretically attractive, whether the activation of complement classical pathway during HIV-1 infection would affect the activation of ADCC remains to be


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determined. We therefore strongly encourage investigators to test this hypothesis. However, to avoid the activation of classical pathway and improve ADCC activity in clinical settings, Fc-defucosylation of anti-HIV-1 antibodies can be used, as aforementioned. 

Competitions Competition is recognized as a strategy that helps HIV-1 escape antiviral immune responses and supports its persistence (Figure 2 and Figure 3). For example, the correlate analysis post-RV144 trial showed that the induction of high plasma anti-HIV-1 IgA antibody titers in vaccine recipients is associated with increased risk of acquiring HIV-1 infection. In part, this is because these antibodies compete with ADCC mediating IgG antibodies for binding to their cognate epitopes on the surface of HIV-1 infected cells66-68, taking into account that the magnitude of ADCC-Abs is very important for optimal mediation of ADCC responses against HIV-1 infected cells. Similarly, the presence of high levels of anti-HIV-1 IgG antibody subclasses that are not capable of activating ADCC responses, such IgG2 and IgG4, can also limit ADCC responses by competing with IgG1 and IgG3 subclasses for binding to the viral envelope glycoproteins that coat HIV-1 infected cells. In another example, the shedding of gp120 from the surface of free HIV-1 particles as well as HIV-1 infected cells could limit the activation of ADCC responses against HIV-1 infected cells. Soluble gp120s could compete with the surface gp120 that coat HIV-1 infected cells for binding to ADCC mediating antibodies, given that soluble gp120 can be detected at high levels in tissues and body fluid samples including blood124,125. Likewise, the shedding of CD16 from the surface of NK cells and other immune cells could also limit ADCC responses against HIV-1 infected



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cells, because these soluble CD16s can act as competitors with the membrane CD16 for the binding to Fc region of ADCC-Abs and also they can limit the activation of ADCC-cells126-128. 

Hiding HIV-1 Epitopes That Activate ADCC Responses It is now well-appreciated that ADCC responses can be mediated by anti-HIV1 antibodies that recognize specific epitopes on the envelope glycoproteins mainly (Figure 1, 2, 4, and 5)129, 130, taking into account that the exposure of these epitopes to ADCC-Abs depends on the viral Env conformation. The HIV1 Env is the viral molecule that is responsible for the viral entry into the target cells. HIV-1 Env is composed of six subunits; three surface gp120 subunits are non-covalently associated with three transmembrane gp41 subunits. The interaction of HIV-1 Env, namely the gp120, with the primary receptor (CD4 molecule) on target cells triggers major conformational changes in HIV-1 Env such as formation of the co-receptor binding site (CoRBS) and bridging sheet, movement of variable loops (V1/V2 and V3), and exposure of the transmembrane gp41 helical hepated repeat (HR1)71, 133. The interaction of host co-receptor (CCR5 or CXCR4) with the CoRBS on CD4-bound HIV-1 gp120 triggers additional conformational changes in the transmembrane gp41 leading to the formation of six-helix bundle that ultimately results in viral fusion with the cellular membrane of the target cell. It is of particular importance to realize that HIV-1 Env is a metastable molecule that transits from the closed conformation (also known as state 1) to its partially opened conformation (also known as state 2) to its final opened conformation (also known as state 3)71. It is worthy to mention that unbound viral Envs of most HIV-1 isolates are in the closed conformation state (state 1), this seems to provide them a protection


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against antibodies attack during natural HIV-1 infection. This is because most of induced ADCC-Abs in HIV-1 infection are non-neutralizing antibodies that target specific epitopes on the viral Env in its opened state 3. Indeed, several studies have demonstrated that binding of CD4 molecule to HIV-1 gp120 is essential to expose such epitopes (e.g. cluster A epitopes; of these epitopes are the highly conserved non-neutralizing A32 and C11 epitopes on gp120, reviewed in

132)

to ADCC-Abs133-136. Noteworthy, mush is known about A32

epitope than C11 epitope132. The importance of the A32 epitope is indicated by: (i) the ability of Fab fragments specific to A32 epitope to inhibit ADCC activity mediated by ADCC-Abs that target this epitope in plasma of chronically HIV1 infected patients137; (ii) the isolation of A32-like monoclonal antibodies from HIV-1 patients with a capability of mediating potent ADCC activity138; (iii) the recognition of C1 peptides by polyclonal antibodies obtained from HIV-1 infected patients that mediate ADCC activity. HIV-1 can minimize the exposure of such epitopes and avoid ADCC responses by down-regulating the expression of CD4 molecules by its Nef and Vpu proteins, as well as abrogating the function of host bone marrow stromal antigen 2 (BST2), which anchors nascent envelope-containing virions at the surface of HIV-1 infected cells, by its Vpu protein (Figure 4)129, 130, 139-142. Hence, exposing such epitopes to ADCC-Abs using CD4mc was suggested as a strategy to enhance ADCC responses143, 144. Intriguingly, other studies indicated that binding of HIV-1 envelope to CD4mc should be coupled with the binding to co-receptors to expose such epitopes (Figure 5)133. Furthermore, targeting viral proteins (Nef and Vpu) and/or antagonizing their mediated functions are strongly suggested to control HIV-1 infection, at least, by means of enhancing ADCC responses. In addition, it is



Page 34 of 78

essential to realize that there are other important factors that can prevent the binding of ADCC-Abs to HIV-1 infected cells such as heavy glycosylation (glycan shield)145, extreme hypervariability90, and limited number of HIV-1 glycoproteins on the surface of infected cells139. 

Latency It is well-recognized that latently HIV-1 infected cells represent a major barrier to completely eradicate the virus. At least, this is because the viruses in these latently infected cells are transcriptionally silent, and thus cannot be recognized by the immune system including ADCC responses (Figure 2). Fortunately, recent investigations have indicated that ADCC could play a critical role in HIV-1 latent reservoirs elimination after sufficient reactivation by anti-latency agents and expression of specific epitopes on these reactivated cells by CD4mc146, 147. This may indicate that the combination of CD4-mc with anti-latency agents may provide an effective strategy to eliminate latent HIV-1 reservoirs by ADCC responses. Of note, the elimination of HIV-1 latent reservoirs after reactivation is essential for strategies that aim at eradicating the virus via "shock and kill" strategy or at achieving a sterile cure. This is mainly because the failure of immune responses to eliminate these reactivated latently infected cells may enable them return back to the latency state, and thus reconstituting the HIV-1 reservoir148. Unfortunately, some studies have reported that certain latencyreversing agents have adverse impacts on the function of NK cells, the principal effector cells involved in ADCC responses, suggesting caution when using such agents, especially when considering ADCC as a strategy to control or eradicate the virus149, 150.



Antiretroviral Therapy 34 ACS Paragon Plus Environment

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It has been recently revealed that ART influences ADCC responses during HIV1 infection. In one longitudinal study, Madhavi and colleagues151,

152

have

shown that HIV-1 infected individuals receiving ART exhibit a significant decline in ADCC-Abs, indicating that ART has negative impacts on ADCCAbs. In line with these data, other studies by Jensen and colleagues153,

154

confirmed these findings; however, they found that initiating ART during the early phase of HIV-1 infection has positive impacts on natural killer cell cytotoxic functions, indicating that ART has negative impacts on ADCC-Abs and positive impacts on ADCC-cells, namely NK cells. However, the reduction of ADCC-Abs during ART can be compensated by the passive administration of ADCC-Abs. This, in turn, can enhance the total ADCC responses, especially when ART is initiated very early after HIV-1 infection. On the other hand, there are several hurdles that can impede the therapeutic success of passive anti-HIV-1 antibodies administration strategy that include the: (i) pre-existence and/or emergence of HIV-1 resistant mutants (mentioned above); (ii) cellto-cell HIV-1 dissemination; and (iii) maintenance of sufficient antibody titers to achieve optimal viral inhibition and to limit emergence of escape mutants, these hurdles were recently reviewed by us elsewhere6. Taken together, when all these relevant hurdles are taken into consideration, the success rate of harnessing ADCC responses as a therapeutic approach for HIV-1 infection will be increased (Figure 2).



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CONCLUSIONS AND FUTURE DIRECTIONS To some extent it is evident that ADCC responses may play a critical role in controlling HIV-1 infection. As such, harnessing ADCC responses to control HIV-1 infection is suggested. Passive administration of combined bNAbs, bi- or tri-specific bNAbs capable of activating ADCC responses (Figure 6) could be implicated to fulfill this purpose. Especially, because this approach will target both the free HIV-1 particles as well as the HIV-1 infected cells, a strategy to “kill two birds with one stone”. Furthermore, we suggest administering such antibodies as early as possible during the course of HIV-1 infection to achieve optimal ADCC activation, because the level of immune dysfunction (defects in effector cells function) will be much less established as compared to the later disease stages. Furthermore, the very early viral control could convert the clinical status of HIV-1 infection to a controller state (functional cure). However, we cannot rule out the benefit of administration of such antibodies during the chronic or even the AIDS phases of HIV-1 infection. Noteworthy that restoring the function of defective effector cells (NK cells) using MMPs inhibitors or application of certain cytokines before administering bNAbs to HIV-1 patients may also benefit them in this context. Of note, to selectively activate ADCC responses using this approach, but not the complement classical pathway (which could limit the optimal activation of ADCC responses by means of competition and negatively effecting ADCC-cells, i.e., NK cells), glycosylation (hypo-fucosylation/defucosylation) of the Fc region of these antibodies is suggested.


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ABBREVIATIONS ADCC, antibody-dependent cellular cytotoxicity; ADCC-Abs, antibodies capable of activating ADCC responses; ADCC-cells, effector immune cells involved in ADCC activation; AIDS, acquired immunodeficiency syndrome; Ig, immunoglobulin; ART, antiretroviral therapy; bNAbs, broadly neutralizing anti-HIV-1 antibodies; BST2, bone marrow stromal antigen 2; CCR5, ; CD, cluster of differentiation; CD4BS, CD4binding site; CD4mc, CD4-memitic compounds; CoRBS, co-receptor binding site; 51Cr,

Chromium; CXCR4, ; Env, envelop; Fab, antigen-binding fragment; Fc,

crystallizable fragment; FcR, Fc receptor; GTL, FcγR, Fc-gamma receptor; GranToxiLux; gp120, glycoprotein 120; gp41, glycoprotein 41; HIV-1, human immunodeficiency virus type 1; HLA, human leukocyte antigen; IFN- γ, Interferon-γ; IL, Interluekin; MMPs, matrix metalloproteinases; NAbs, neutralizing anti-HIV-1 antibodies; Nef, negative factor; NK, natural killer; nNAbs, non-neutralizing antiHIV1 antibodies; RFADCC, rapid and fluorometric ADCC; SHIV, simian-HIV; SIV, simian immunodeficiency virus; Vpu, viral protein U.

AUTHOR INFORMATION Nizar Mohammad Abuharfeil1*, Mahmoud Mohammad Yaseen2*#, Fawzi M. Alsheyab1 1Department

of Applied Biological Sciences, College of Science and Arts, Jordan

University of Science and Technology, Irbid, 22110, Jordan 2Department

of Medical Laboratory Sciences, Faculty of Applied Medical Sciences,

Jordan University of Science and Technology, Irbid, 22110. Jordan



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CONFLICT OF INTEREST All authors declare that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ACKNOWLEDGMENT This work was supported by the Deanship of Research at Jordan University of Science and Technology grant number 365/2017.


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FIGURES AND LEGENDS Figure 1

Legend. ADCC mediated activity against HIV-1 infected or Env-coated cells. In fact, for ADCC activity to be mediated, three participants must be coexisted: first, the target cell (HIV-1 infected or Env coated cells); Second, the specific anti-HIV1 antibodies capable of mediating ADCC activity (ADCC-Ab); Third, the effector cell (e.g., NK cell). As a result of viral replication in HIV-1 infected cells, viral Env glycoproteins (closed Env conformation, state 1) are produced and trafficked to the cell membrane to initiate the assembly/budding process. These closed Env glycoproteins could bind to the CD4 molecule present on the cells surface leading to conformational changes in the viral Env glycoproteins resulting in an open conformation Env state 3 that expose certain hidden epitopes to ADCC-Abs (most likely these antibodies are non-neutralizing because neutralizing antibodies can recognize HIV-1 Env in its closed state). This event sensitizes the target cell to killing by NK cell-mediated ADCC activation (degranulation and cytolysis of target cell). Of note, this scenario of ADCC activation is likely initiated in the absence of


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Nef and Vpu viral proteins (which can downregulate the CD4 molecule at the surface of infected cells155, 156, as seen in the case of defective HIV-1 (lacking Nef and or Vpu) infection or coating cells with Env.



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Figure 2

Legend: Factors that influence ADCC activation during natural HIV-1 infection. For ADCC responses to be mediated against HIV-1 infected cells there should be functional ADCC mediating effector (e.g. natural killer, NK) cells and anti-HIV-1 antibodies (ADCC-Abs) capable of binding to specific exposed viral epitopes in the case of non-neutralizing antibodies (E-Gp120) expressed on the surface of HIV-1 infected cells as seen in the case (1). The presence of functional NK cells and ADCC-Abs but not capable of binding to non-exposed viral epitopes (N-Gp120) in the case (2) prevent the activation of ADCC. Cases 3 and 4 represent dysfunctional NK cells that cannot proceed to activate ADCC responses. Competition between the soluble form of FcγR (sFcγR) and the membrane bound form FcγR for binding to Fc portion of ADCC-Abs could limit the ADCC activation as illustrated in case (5). Likewise, are the cases (6, 7, and 8), for case (6) non-ADCC-Abs will compete


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with ADCC-Abs for binding to E-Gp120. In case (7), the presence of soluble Gp120 will compete with E-Gp120 for binding to ADCC-Abs. While for case (8), the presence of complement classical activating complex C1 competes with FcγR for binding to Fc portion of ADCC-Abs. In case (9) the absence of viral proteins expression in latently HIV-1 infected cells prevents ADCC activation against these cells. Finally, the absence of ADCC-Abs will also prevent ADCC responses activation as in the case (10).



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Figure 3

Legend: Competition between complement classical pathway and ADCC. A) The presence of IgG1/3 antibodies along with the complement activating proteins (C1) limit the activation of ADCC by means of competition, since C1s will compete with FcγRs for binding to the Fc portions of ADCC mediating antibodies. B) Inhibition of C1 enhances ADCC activation. C) Alternatively, afucosylation or hypofucosylation of Fc region of anti-HIV-1 antibodies selectively enhances ADCC responses by enhancing the antibody binding to FcγR expressed on effector cells (i.e., NK cells), but not (C1), thus limiting the activation of complement classical pathway.


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Figure 4

Legend. Mechanisms of ADCC immune response evasion by HIV-1 via Nef and Vpu155,156. HIV-1 has unique multifunctional accessory proteins Nef and Vpu. One mechanism that the virus uses to evade ADCC activity by these proteins is the downregulation of CD4 molecule on the surface of target cells, in which its interaction with Env results in sensitizing target cell by ADCC-Abs to NK cell killing by the means of ADCC activity. Nef can mediate the internalization (endocytosis) of the surface CD4 molecules and drive them to degradation by lysosomes. While Vpu drives the degradation of newly synthesized CD4 through the proteasome pathway, thereby resulting in downregulation of CD4 molecule on the surface of target cells. Furthermore, HIV Vpu can counteract the function of restriction factor BST-2, which anchors nascent virions at the surface of target cells and, thus preventing the accumulation of nascent virions on the surface of infected cells, which makes them less vulnerable to recognition by ADCC-Abs and killing by NK cells.



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Figure 5

Legend. The effect of CD4mc and co-receptor binding site (CoRBS) antibodies on sensitizing target cells infected with competent HIV-1 (have Nef and Vpu proteins) to NK cell-mediated ADCC activity upon binding to ADCC-Abs. First, Nef and Vpu can downregulate the CD4 expression on target cells155,

156.

This event will prevent the

binding of the viral Env to CD4 and thus preventing the transition of Env from the closed conformation state to the opened conformation state. This will hide specific epitopes recognized by ADCC-Abs. As a consequence, this will protect target cells from NK cell-mediated ADCC activity. However, application of CD4 memitic compounds (CD4mc) results in transition of the viral Env from the closed state (state 1) to a partially opened state (state 2), this will expose CoRBS epitopes to CoRBS-Abs resulting in transition of the Env from the state 2 to the fully opened state (state 3). As a result, specific epitopes are exposed to non-neutralizing ADCC-Abs leading to activation of NK cell-mediated ADCC activity and killing of target cell.


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Figure 6

Legend: A proposed model describing passive administration of defucosylation or hypo-fucosylated Fc broadly neutralizing anti-HIV-1 antibodies (BNAbs) capable of activating ADCC responses as a strategy to control HIV-1 infection. (1) Testing the susceptibility of autologous HIV-1 isolates to the tested BNAbs helps determine the optimal combination of antibodies and avoid HIV-1 resistance (for more details about BNAbs combinations see reference157. (2) Measuring the baseline viral load and CD4+ T cells count helps determine the dose of BNAbs required to be passively administered and the functionality of immune responses, respectively.



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Harnessing Antibody-Dependent Cellular Cytotoxicity To Control HIV

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