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Chapter 10
Preclinical Assessment of Glycoconjugate Vaccines Ingrid L. Scully, Kena A. Swanson, Isis Kanevsky, A. Krishna Prasad, and Annaliesa S. Anderson* Pfizer Vaccines Research and Development, 401 N. Middletown Rd., Pearl River, New York 10965, United States *E-mail:
[email protected].
Glycoconjugate vaccines are complex biological entities made from combining a polysaccharide with a carrier protein. The resulting vaccine must retain the biological appearance of the pathogen’s polysaccharide to drive an immune response that can recognize and facilitate killing of the pathogen. Polysaccharde structures vary across serotypes and even among strains within a serotype. Thus, there is not a single conjugation approach that can be applied to all polysaccharide conjugate vaccines, and the ability of candidate vaccines to elicit functional immune responses is often empirically determined. To identify an optimally immunogenic vaccine candidate, both in vivo and in vitro models can be useful. Important considerations for preclinical glycoconjugate vaccine evaluation include: (a) selection of appropriate in vivo models to measure immunogenicity, (b) development of in vitro assays that measure immune responses that facilitate killing of the pathogen, and (c) identification of glycoconjugate features/epitopes that are critical for eliciting functional immune responses. These considerations are illustrated with case studies.
© 2018 American Chemical Society Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Introduction Capsular polysaccharides (CP), expressed on the cell surface of most bacterial pathogens, have evolved to serve as an immune evasion mechanism, effectively masking the bacterial cell from recognition by the host innate immune system. When presented in the context of the whole bacterium they are poorly immunogenic. It is the repetitive and dense antigenic structure of these molecules that convert these structures from the bacteria’s armour to their Achilles heel, as vaccines made from these structures have proven highly effective at generating immune responses that can facilitate the killing of the pathogen upon entry into the blood stream. CP alone vaccines have been licensed since the 1970s for the prevention of disease caused by Neisseria meningitidis, eg quadrivalent meningococcal polysaccharide vaccine (MSPV4), and Streptococcus pneumoniae, eg pneumococcal polysaccharide vaccine – 23 valent (PPSV23). However, unconjugated capsular polysaccharide vaccines have been shown to be poorly immunogenic in infants, and may even induce blunted booster responses (1, 2), limiting their application in preventing disease in infants and young children, the age groups that are at greatest risk for poor outcomes of infectious disease. Early in the 20th century, as polysaccharides from S. pneumoniae were first being discovered, Oswald Avery and colleagues at the Rockefeller Institute for Medical Research used preclinical studies to demonstrate that purified polysaccharides were unable to generate protective immune responses in animal experiments, whereas vaccines made from whole bacteria could induce serotype-specific responses and confer protection in vaccinated animals. This observation led them to conjugate the polysaccharides to proteins after which they observed improved protection in animal models (3). Protection observed in humans but not animals with unconjugated CP vaccines could be due to the fact that humans have prior exposure to the pathogen and thus CP vaccination elicits an anamnestic response. In contrast, animals, especially those housed in specific pathogen free facilities, commonly lack any pre-existing immunity, and thus cannot mount memory responses to the CP vaccine. The immunization schedules used in early animal experiments did not address this concept. Likewise, for young children (under two years of age) who do not have fully developed immune systems and have not had the opportunity to build natural immunity, CP vaccines alone were not effective (4). The addition of the protein carrier to the CP shifts the immune response from a T cell-independent response to a T cell-dependent response, resulting in both enhanced immunogenicity, especially in young children under the age of two, and enhanced immunological memory (5). Although the concept of the glycoconjugate (CP conjugated to a protein carrier) is similar across vaccines, the details of the immune response to each glycoconjugate vaccine is unique, and requires preclinical evaluation. Predictive in silico modeling techniques do not yet exist that can accurately determine which polysaccharide conjugate attributes, such as size, substituents, or degree of cross-linking, result in functional, protective immune responses. As described in other chapters in this volume, many chemical approaches can be tried to design and develop a glycoconjugate. Each approach may or may not impact the presence and/or accessibility of important immunogenic epitopes on the 230 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
glycoconjugate. In examining the variables associated with conjugate production (Figure 1), the preclinical assessments can broadly distinguish whether vaccines can still induce antibodies that recognize and facilitate killing of the pathogen.
Figure 1. Critical quality attributes for CP-conjugate vaccine design and the role of preclinical assessments.
Ideally, both an animal model that recapitulates human disease and an in vitro assay that can accurately measure functional antibody responses would be available to test candidate conjugate vaccines. In practice, the perfect pair of animal model that accurately predicts clinical efficacy and an in vitro assay that perfectly monitors the functional immune response rarely exists. This truism requires that preclinical animal model data be married with in vitro assay data to identify the appropriate path forward for vaccine development. In practice, this approach can be used to demonstrate broad differences between vaccine formulations. However, the approach is marred by the intrinsic difference between humans and preclinical animal models, most notably the inability of many animals to differentiate between CP and CP-conjugate vaccines. Rodent and rabbit models are often selected for initial assessment of candidate vaccine immunogenicity, due to cost and handling considerations. While small animal models are powerful tools and can rapidly screen large numbers of candidates, they do have their limitations, as highlighted in the following case studies. Nonhuman primates are often used to evaluate the immunogenicity of more advanced candidates, as they are more closely phylogenetically related to humans. However, even nonhuman primates cannot always completely predict immune responses in humans, especially in terms of the magnitude of responses.
231 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Some considerations for the use of animal models are: • • • •
•
Is the dose and route of immunization appropriate for the animal model? Is the age of the animals appropriate? Immune responses vary in young vs older mammals, due to the development of the immune system. Is the species appropriate? Does this species respond to the glycoconjugate? Does the species become infected with the pathogen from which the glycoconjugate is derived? How similar is the infectious process in the preclinical animal model and in the human vaccine target population? If challenge models are to be used, are clinically relevant strain(s) being tested in the model or conventional lab-adapted strains?
The impact of animal model selection is illustrated in the case studies presented later in this chapter. As highlighted above, selection of appropriate in vitro assays to complement preclinical in vivo models is important for successful development of candidate conjugate vaccines. Levels of total immunoglobulin generated by conjugate vaccines are often measured in ligand binding assays, such as enzyme-linked immunosorbent assays (ELISAs) or Luminex-based immunoassays (LIAs). The concept of ELISA and LIA is similar. In the case of ELISAs, the readout is the product of an enzymatic reaction that is proportional to the amount of antibody bound; while in the case of LIAs, the readout is fluorescence that is proportional to the amount of antibody bound to the target antigen. LIAs are powerful as the technology allows for assessment of antibody responses to multiple antigens in a single sample reaction, termed multiplexing. These assays can be run in different formats, such as direct-binding LIA (dLIA), capture LIA (caLIA), or competitive LIA (cLIA). All of these have the benefit of providing information on the magnitude of the antibody response. They do not provide direct assessment of the functional activity (quality) of the antibodies. The prototypical serological assays used for evaluation of functional antibody responses are the serum bactericidal assay (SBA) and the opsonophagocytic killing assay (OPA/OPK). The SBA measures the ability of antibodies to kill bacteria by the classical and alternate complement pathways: specifically antibodies bind to their target and recruit complement, which is activated and induces the membrane attack complex to kill the pathogen. The SBA measures this by mixing of target bacteria with serial dilutions of test antibody containing serum and a source of complement. After an incubation period, aliquots of the assay reaction are plated, and the surviving bacteria are enumerated. The reciprocal of the serum dilution where half of the bacteria survive, compared to an input value, is reported as the SBA titer. As the SBA measures direct bactericidal activity of serum mixed with exogenous complement, the source of complement can have a strong outcome on the titers obtained. Therefore, the species source of complement must be carefully considered (e.g. human versus animal) and complement must be prescreened to ensure nonspecific background killing activity is not present. The OPA measures the ability of antibodies to induce phagocytic killing of the pathogen. It is similar to the SBA, except for the addition of phagocytes, 232 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
which engulf and kill the pathogen in the presence of functional antibodies, as opposed to direct bactericidal activity monitored in the SBA. Like the SBA, bacteria are mixed with serum in the OPA, allowing antibody to bind to and “opsonize” the bacteria. A source of complement and phagocytes are then added to the bacteria-serum mixture, and the reaction is allowed to proceed for a period of time, generally 30 minutes to 2 hours. The phagocytes included in the OPA should be relevant for the pathogen being tested. A commonly used phagocytic cell source is HL-60 cells, a human promyelocytic cell line that can be differentiated into neutrophil-like cells. Surviving bacteria are enumerated at the end of the assay period by plating aliquots of the reaction mixture onto agar plates or onto filter plates (microcolony assay). Colonies are counted the next day, either through direct visualization or through staining and counting by means of an immunospot analyzer. OPA titers are generally reported as the inverse of the serum dilution that results in killing of 50% of the input bacteria. By using bacterial strains that have been genetically modified to contain antibiotic resistance genes, the OPA can be multiplexed. It should be noted that the term OPA can also be used to refer to a bacterial uptake assay, as opposed to a true assessment of bacterial killing; for this reason some members of the field prefer the term OPK to distinguish the killing assay from the uptake assay. As OPAs measure serum-induced bacterial killing, they are widely used in the assessment of glycoconjugate vaccine responses. It is critical to note that binding antibody levels are not always indicative of a functional serological response. There are many clinical examples where polysaccharide antigens have induced high binding responses without subsequently demonstrating functional responses. For example, binding responses to pneumococcal serotype 19A following immunization with serotype 19F polysaccharide do not correlate with OPA responses to 19A (6). Determining the quality of the antibody response is important when assessing glycoconjugate vaccine candidates. Therefore, it is critical to incorporate in vitro assays that can assess both binding and functional antibody responses. Case studies are presented in this chapter, to highlight the following considerations: • • •
Selection of appropriate models to evaluate immunogenicity of conjugate variants. Development of in vitro models that assess functional immune responses, related to interruption of microbial pathogenesis. Identification of critical epitopes that must be preserved to ensure a conjugate elicits the desired functional response.
Preclinical Evaluation of Conjugate Vaccines – Haemophilus influenzae b Before effective vaccines became available, Haemophilus influenzae type b (Hib) was the most prevalent cause of invasive bacterial infection in children under 5, a rate of ~1 in 250 live births; approximately 2/3 of cases were in children under 18 months of age. Over half of Hib invasive disease in the prevaccine era presented 233 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
as meningitis, with a 3-6% case fatality rate, despite antimicrobial therapy. Up to 30% of survivors of Hib meningitis experienced neurologic sequelae and/or hearing loss (7). Natural history studies of Hib infection in the 1930s (8) noted that the risk of Hib-induced meningitis was greatest in infants aged 9-12 months, representing the time between when antibody titers transferred from the mother waned and when the child gained their own protective antibodies. It was later shown in the 1960s that the protective Hib antibodies generated in older children and adults reacted to the Hib capsular polysaccharide. These findings highlighted the need to identify a method of generating protective antibodies to pathogen capsular polysaccharides in infants and young children. Seminal work by John Robbins, Rachel Schneerson and colleagues showed that free polysaccharide was poorly immunogenic in mice and nonhuman primates, while glycoconjugates induced robust protective immune responses (9, 10) (Figure 2).
Figure 2. Responses of Juvenile Rhesus Macaques to Polysaccharide vs Conjugate Vaccination. Juvenile rhesus macaques were immunized three times subcutaneously with 50 mcg of Hib-tetanus toxoid conjugate (Hib-TT), Hib alone or TT alone, and anti-Hib antibody titers were measured by ELISA. (Adapted from (10). Copyright 1984, American Society for Microbiology). Likewise, immunogenicity of glycoconjugates in human adults was not predictive of responses in infants (11, 12). Infants and young children do not have fully developed immune systems, and so the immune responses elicited in adults are not always replicated in infants and young children. Importantly, it was noted that not all proteins and conjugation methods were able to act as efficient polysaccharide carriers to elicit functional antibody responses. Porter Anderson and colleagues found that cross-reactive material 197 (CRM197), a genetically detoxified variant of diphtheria toxin, was an effective carrier protein to induce high-titer antibody responses. This was reduced to practice by David Smith and 234 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
colleagues at Praxis Biologics, resulting in a successful vaccine against Hib that could protect infants and young children from invasive Hib disease (7). For this lifesaving work, John Robbins, Rachel Schneerson, Porter Anderson and David Smith received the Lasker Award in 1996. Today, vaccine-mediated protection against Hib prevents Hib-mediated meningitis in infants and young children.
Of Mice and Men: A Pneumococcal Case Study Streptococcus pneumoniae is a major cause of pneumonia, bacteremia, and acute otitis media and is associated with significant morbidity and mortality worldwide in children less than 2 years of age and elderly adults (13, 14). S. pneumoniae is a gram-positive bacterium containing a capsular polysaccharide (CP). Composition of the CP varies across the more than 90 known pneumococcal serotypes and is the major component in currently licensed vaccines against the prevalent disease-causing serotypes. The 23-valent CP alone vaccine (PPSV23, PneumoVax®), is licensed globally for the prevention of pnemococcal disease in adults, and two glycoconjugate vaccines, the13-valent PS conjugate vaccine (PCV13, Prevnar13®), licensed globally for use in all ages, and another 10-valent vaccine, PCV10, licensed outside the US for use in children 6 months to 5 years of age (15–18). Conjugation of the CPs to the protein carrier CRM197 (non-toxic mutant form of diphtheria toxin) in PCV13 drives a T-cell-dependent immune response and subsequent improved memory B-cell and antibody response, in contrast to the T-independent response elicited by unconjugated vaccines whose protection has been observed to wane following vaccination (19). The power of a conjugate vaccine to elicit protective immune responses was demonstrated in 1929, when Avery and Goebel showed that immunization with a bacterial polysaccharide conjugated to a protein carrier induced robust protective antibody responses against the bacterium (20). In 1931, Avery and Goebel expanded their work with a Streptococcus pneumoniae serotype III polysaccharide conjugated to a protein carrier, which induced anti-serotype III antibodies in rabbits, and these antibodies, when passively transferred to mice, could protect mice from lethal infection with S. pneumonia (21).Development of pneumococcal PS conjugate vaccines has relied on many animal models for evaluation of immunogenicity and protection against bacterial challenge or colonization, e.g. mice, rabbits, nonhuman primates, infant rat bacteremia model, and the chinchilla otitis media model. Mice are routinely used (adult, infant, and aged mice) for assessment of conjugate immunogenicity in part due to ease of access to animals and immunological reagents. However, in some cases, mice do not mount immune responses against all S. pneumoniae serotypes, making rabbits a reliable model to discriminate between conjugated and unconjugated PSs. Some reports suggest responses to PS and PS-conjugate vaccines in mice and rabbits may predict efficacy in humans (22–24). As described earlier, similar to humans, young mice do not respond well to unconjugated PS vaccines, and priming with unconjugated PS can result in a suboptimal response following subsequent PS conjugate boosting (3, 25). 235 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
A comparison of responses in humans to those observed in mice has not been directly tested. However, a recent study by Caro-Aguilar et al found varying responses to a 15-valent PS conjugate vaccine (PCV15) depending on the strain of mouse and route of immunization (26). This vaccine is currently in clinical development by Merck & Co. IgG responses were observed for all serotypes in all mouse strains tested: two inbred (Balb/c, Swiss Webster) and two outbred (C3H, and CD1). Serotypes 6B, 23F and 33F were less immunogenic in all strains except CD1 mice. Previous studies have shown that some serotypes, including 23F (27) and 33F (26), are poorly immunogenic in mice. CD1 mice, being outbred, may have sufficient genetic diversity to engender an improved immune response to a more diverse array of PS serotypes compared to inbred mice but further study would be warranted. CD1 mice were also indifferent to an intramuscular (IM) or intraperitoneal (IP) vaccination route, showing similar antibody responses compared to other mouse strains that displayed increased responses with IP vaccination. Because of their genetic relatedness to humans, nonhuman primates (NHPs) have been used to assess PS conjugate vaccine responses. Recently published observations in adult cynomolgus macaques suggest NHPs, like human adults, show a superior immune response to conjugated PS vs. PS alone. NHPs vaccinated with a 7-valent PS conjugate vaccine and boosted five years later with either a 23-valent PS vaccine (PPSV23) or 13-valent PS conjugate vaccine (13vPnC) displayed an increased breadth and diversity of the antigen-specific memory B cell response against the 13vPnC booster compared to animals that received 23vPS booster (28). An infant macaque immunogenicity model has been used to evaluate whether it is predictive of responses in human infants. Infant macaques possess immature immune systems similar to humans and are susceptible to some infectious pathogens making them useful to test in the context of vaccine immunogenicity and efficacy (29). For pneumococcal conjugate vaccines, infant rhesus monkeys have been used to assess potential immunological interference with increasing valency in conjugate vaccines. As the disease epidemiology has evolved post-introduction of PPV23 and PCV13, non-vaccine serotypes are emerging (30), suggesting more broadly protective vaccines may be needed. The PCV15 vaccine in clinical development is attempting to address the expanding medical need by including two new disease-causing serotypes (22F and 33F) together with the existing 13 serotypes in PCV13. Prior to studies in humans, antibody responses to PCV15 compared to Prevnar 7 were compared in infant rhesus macaques. In infant monkeys 2 to 3 months of age, IgG and functional opsonophagocytic (OPA, described earlier) antibody responses following three doses of either PCV15 or Prevnar 7 (7-valent PS conjugate vaccine) were comparable for the 7 serotypes common to both vaccines suggesting no immunological interference with PCV15 (31). Post-vaccination responses to PCV15 were >10-fold higher than baseline for the 8 additional serotypes. The PCV15 vaccine has been tested in human adults, toddlers, and infants, as described in Chapter 2 in this volume (32). In contrast to data in infant rhesus monkeys, IgG responses in human infants showed reduced serum IgG levels for serotypes 6A and 19A. These observations suggest the infant rhesus model insufficiently predicts signs of immunological 236 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
interference or other differences in immunity in human infants when comparing pneumococcal conjugate vaccines with increasing numbers of serotypes. It is unknown whether this would apply to other glycoconjugate vaccines or alternative vaccine platforms. Immunogenicity studies can also be complemented with challenge models to further assess the functionality of immune responses. In the case of pneumococcal vaccines, the infant rat passive protection model has been used extensively to assess functional serological responses. Pneumonia models in rats and nonhuman primates have been employed to assess preclinical efficacy of vaccine candidates. In addition, a chinchilla otitis media model is sometimes employed to assess preclinical responses. There are many parallels with the immune response in rodents and NHPs to those observed in humans. Mice and rabbits are simple models that allow for preclinical screening of PS conjugate immunogenicity. In cases where mice do not mount an immune response against a specific S. pneumoniae serotype, it is important to consider tailoring the PS conjugate dose and/or inclusion of adjuvant to overcome these apparent challenges. Sufficient evidence supports the use of adult NHPs for characterization of the antibody and cellular immune response. With the disease landscape evolving and some evidence for the emergence of antibiotic-resistant S. pneumoniae strains, these underscore the continuing importance of improving animal models in support of advancing new vaccines against pneumococcal disease.
A Tale of Two O-Acetyls: A Case Study of Staphylococcus aureus and Neisseria meningitidis Glycoconjugates Staphylococcus aureus is carried asymptomatically in the nares of 20-50% of the general population (33). Colonization increases the risk of infection, ranging from relatively mild skin infections, such as impetigo, to life-threatening invasive disease. S. aureus is recognized as a leading cause of morbidity and mortality in both healthcare-associated and community settings. In particular, infections in surgical patients carry high mortality rates and survivors of S. aureus surgical infections require an additional 13-17 days in the hospital, significantly increasing healthcare costs (34). The burden of S. aureus disease is exacerbated by the emergence of S. aureus isolates that are resistant to new classes of antibiotics, highlighting the need for alternative approaches such as a prophylactic vaccine. One of the S. aureus vaccines that was not successful in the clinic was comprised of capsular polysaccharide conjugates. Capsular polysaccharides help bacteria evade immune-mediated killing through inhibiting phagocytosis (35, 36). Vaccine-induced antibodies against capsular polysaccharides can overcome this virulence mechanism by enabling the organism to be opsonized and subsequently phagocytosed. All invasive human S. aureus isolates encode the genes required to express either type 5 or type 8 capsule (denoted CP5 and CP8, respectively), and most adults have anticapsular antibodies, demonstrating that the capsule is expressed in vivo. Due to its highly repetitive nature, capsular antigens have high epitope density, allowing multiple antibodies to bind, and thus are attractive 237 Prasad; Carbohydrate-Based Vaccines: From Concept to Clinic ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
candidates for prophylactic vaccines. Bacterial polysaccharides often contain an array of substituents, such as O-acetyl, phosphate, and sialic acid (37), which may constitute an important part of the immunodominant epitopes. Both S. aureus CP5 and CP8 are comprised of 2-acetamido-2-deoxy-Dmannuronic acid (ManNAcA), 2-acetamido-2-deoxy-L-fucose (L-FucNAc), and 2-acetamido-2-deoxy-D-fucose (Figure 3). Both capsules are O-acetylated and differ in the stereochemical nature glycosidic linkages between the sugars and the site of O-acetylation. The sites of O-acetylation are the 3′OH moiety of L-FucNAc for CP5 and 4′OH substituent of ManNAcA for CP5 (38).
Figure 3. Structures of staphylococcal polysaccharide serotypes 5 and 8.
The presence of O-acetyl groups in capsular polysaccharides of pathogenic bacteria including Escherichia coli K1 (39), N. meningitidis groups A (40), C, H, I, K, W, and Y, Salmonella enterica serovar Typhi (41) Paratyphi A (42), S. aureus serotypes 5 and 8 (38, 41) S. pneumoniae type 9V (43) and group B streptococcus serotypes Ia, Ib, II, III, V and VI (44) has been widely observed. However, the role of O-acetylation in the immunogenicity and pathogenicity of microorganisms cannot be generalized (39, 45, 46). In the case of S. aureus, O-acetylation of the capsular polysaccharide was shown to be critical for the elicitation of functional antibodies that can kill the organism in vivo and in vitro (47). S. aureus causes a wide range of disease in a variety of host microenvironments. Therefore the preclinical development of an effective vaccine targeting S. aureus must involve the use of multiple preclinical in vivo models which represent different infection modalities. In the case of the Oacetylation assessment, the end-organ infection model, pyelonephritis, was used. In this model, immunization with de-O-acetylated conjugates were poorly able to reduce bacterial load, while immunization with fully O-acetylated conjugates were able to reduce bacterial load by an additional two logs, a highly significant reduction (p
