A Rapid Blood Test To Determine the Active Status and Duration of

Subscriber access provided by Caltech Library

Article

A Rapid Blood Test to Determine the Active Status and Duration of Acute Viral Infection Tianyu Zheng, Caroline Finn, Christopher Parrett, Kunal Dhume, Ji Hae Hwang, David Sidhom, Tara Strutt, Yuen Yee Li Sip, Karl McKinstry, and Qun Huo ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00137 • Publication Date (Web): 17 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Infectious Diseases is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

A Rapid Blood Test to Determine the Active Status and Duration of Acute Viral Infection Tianyu Zheng a, Caroline Finn b, Christopher J. Parrett b, Kunal Dhume b, Ji Hae Hwang b, David Sidhomb, Tara M. Strutt b, Yuen Yee Li Sip b, Karl K. McKinstry b*, Qun Huo a* a

Department of Chemistry and NanoScience Technology Center, University of Central Florida,

12424 Research Parkway Suite 400, Orlando, FL 32826

b

Burnett School of Biomedical Science, Division of Immunity and Pathogenesis, College of

Medicine, University of Central Florida, 6900 Lake Nona Blvd., Orlando, FL, 32827

*To whom the correspondence may be addressed: [email protected] and [email protected]

The ability to rapidly detect and diagnose acute viral infections is crucial for infectious disease control and management. Serology testing for the presence of virus-elicited antibodies in blood is one of the methods used commonly for clinical diagnosis of viral infections. However, standard serology-based tests have a significant limitation: they cannot easily distinguish active from past, historical infections. As a result, it is difficult to determine whether a patient is currently infected with a virus or not, and on an optimal course of action, based off of positive serology testing responses. Here we report a nanoparticle-enabled blood test that can help overcome this major challenge. The new test is based on the analysis of virus-elicited immunoglobulin G (IgG) antibody present in the protein corona of a gold nanoparticle surface upon mixing the gold nanoparticles with blood sera. Studies conducted on mouse models of

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

influenza A virus infection show that the test gives positive responses only in the presence of a recent acute viral infection, approximately between day 14 to day 21 following the infection, and becomes negative thereafter. When used together with the traditional serology testing, the nanoparticle test can determine clearly whether a positive serology response is due to a recent or historical viral infection. This new blood test can provide critical clinical information needed to optimize further treatment and/or to determine if further quarantining should be continued.

KEYWORDS: Viral Infection Diagnosis, Influenza Virus, Immune Response, Gold Nanoparticle, Dynamic Light Scattering, Biomolecular Corona, Antibody.

Clinical detection and diagnosis of viral infection faces many challenges. Most acute viral infections follow a typical three-stage pathological process: early stage infection including latent incubation and virus shedding; a transition stage when the immune response starts to take place; and the recovery stage with full development of antibody responses and complete clearance of infectious virus from the system. During this evolving process, the viral titer and antibody (Ab) titer reaches its peak value at very different time points. Typically, viral titer reaches the highest during the first week of infection and the Ab titer peaks at around day 21 following initial infection. Because of this sequential development, different diagnostic tests have to be used in clinical diagnosis of viral infections and each has its own limitations and challenges.1 Viral protein detection and RT-PCR-based nucleic acid testing can be used to detect and diagnose early stages of viral infection. However, these tests are only effective when relatively high levels of virus particles are still present in the system. If the patient has already passed the early stage


Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of infection and entered into immune response stage, the viral titer will decrease significantly as a result of clearance by the immune system. Consequently, a negative test result from antigen detection or nucleic acid testing could miss these patients who have progressed beyond the early stages of infection, leading to false negative diagnosis. Similarly, such approaches may display reduced sensitivity against infectious agents that target a specific tissue-site versus those that cause systemic infection resulting in greater amounts of virus and viral antigen to be present in the blood. Serology testing is commonly used to detect virus-specific antibodies that are produced during active immune responses to a viral infection. Despite the recently increased use of RTPCR technique in diagnosis due to their high sensitivity, serology testing remains an essential core element of diagnostic virology: once the patient passes the early stage of infection, virusspecific IgG Ab are the only biomarker left in the blood that can be reliably used for diagnosis. However, serology testing also faces a significant limitation in that it cannot determine whether the positive response is caused by a current, ongoing viral infection or by a past, historical infection (or vaccination) event, due to the long-lived nature of IgG that can persist for years in the circulation.2 For some infectious diseases, the ability to distinguish the two scenarios has critical clinical implications. A relevant example is the recent Zika virus outbreak.3 Because Zika infection is linked to microcephaly and other severe neurodevelopmental complications in the children of infected pregnant women,4 there is a critical need for new diagnostic tests that can determine when a Zika-infected woman is completely cleared of the virus from natural recovery or medical treatment, and thus when pregnancy no longer carries increased risk. Several years ago, our group developed a new biomolecular assay platform using gold nanoparticle (AuNPs) probes combined with dynamic light scattering (DLS) detection.5,6 Named



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

as D2Dx (from diameter to diagnostics) assay, this technique detects biomolecular species and targets by monitoring AuNP aggregate formation upon binding of specific target molecules with AuNP probes using DLS technique. D2Dx is a homogeneous solution, label-free and washingfree assay technique. It is easy to perform and the results are obtained within minutes. Since our initial report of this technique, D2Dx has been used for detection and analysis of a wide range of target analytes including proteins, DNAs, viruses, and toxins.7-15 The fast response time of the D2Dx assay makes this technique especially appealing for diagnostic virology applications. A study reported by Driskell et al.8 demonstrated that the use of this technique for influenza virus particle detection has led to a two orders of magnitude improvement on sensitivity compared to commercially available products. In the last few years, we have focused on using D2Dx assay to study and analyze proteins adsorbed to the AuNP surface from blood as a new approach for biomarker discovery and detection.16-18 Blood contains a large quantity and variety of proteins and other biomolecules. It is known that proteins and some other types of biomolecules from blood can spontaneously adsorb and self-assemble into a thin biomolecular film called a “protein” or “biomolecular corona” on the nanoparticle surface through non-covalent interactions. Both high and low abundance blood proteins have been found in such corona structures.19-23 Because a pathological condition can change the quantity or types of proteins and other relevant biomolecules present in the blood, we hypothesize that by analyzing the composition of the biomolecular corona, it is possible to discover molecular differences between patients versus healthy controls, and such molecular differences may be used as biomarkers for diagnostic applications. During an acute viral infection, immune responses are initiated to contain and clear the threat. Because of the systemic nature of the immune response, the composition of proteins and


Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


other biomolecules in the blood is dramatically altered. In particular, a key event of immune responses against acute viral pathogens is the production and release of virus-specific Ab, especially of the IgG isotype in the blood. We therefore hypothesized that an increased amount of IgG may be present in the AuNP protein/biomolecular corona at time points post-infection when IgG responses are known to be initiated. To investigate this hypothesis, we designed a simple two-step homogeneous solution assay as illustrated in Figure 1 using the D2Dx platform to analyze the relative quantity of IgG present in the AuNP protein corona. In the first step of the assay, a small amount of test serum (2 µL) is mixed with 40 µL of citrate-capped AuNP solution. Following 15-20 min of incubation, a stable protein/biomolecular corona is formed on the AuNP surface. Then in the second step of the assay, a species-specific anti-IgG Ab is added to the assay solution. If IgG is present in the protein corona, the anti-IgG Ab will bind with the IgG present in the serum adsorbed to the AuNP, causing crosslinking and clustering of AuNP, leading to a substantial increase of the average AuNP size that can be readily detected by DLS. A test score, defined as the ratio of the average particle size from the second (D2) versus the first step assay (D1), is used to evaluate the relative quantity of IgG present on the AuNP surface. Using this assay, we investigated the immune response of wild-type C57BL/6 (H-2b) mice following infection with a sublethal dose of the mouse-adapted A/PR8 influenza A virus (IAV). IAV infection generates a robust antibody response that is required for complete viral clearance (which occurs around 14 days post infection). This makes the murine influenza model wellsuited to address the aims of this study. The mice were bled kinetically at different days for serum analysis. From our study, we discovered that the new nanoparticle test gives positive results only during a well-defined window following viral infection, and then returns back to negative. This window appears to be from the period between 14 and 21 days post-infection,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

correlating with the period immediately following viral clearance and the resolution of the antiviral immune response. The nanoparticle test outcome is in stark contrast to the traditional enzyme-linked immunosorbent assay (ELISA) serological testing for virus-specific antibody, which remained positive long after the infection is cleared. Importantly, we show that the nanoparticle test detects immune responses from both a primary and secondary, heterosubtypic challenge that mirrors pandemic infection. The test can also distinguish the different levels of immune responses from wild type mice with intact immune functions and mice with compromised humoral immunity against IAV.

EXPERIMENTAL SECTIONS Animal Subject Research and Protection. All experimental animal procedures were approved and conducted in accordance with the University of Central Florida’s Animal Care and Use Committee (IACUC) guidelines.

Virus infection of mice and blood collection. 8-12-week-old wild-type C57BL/6 mice or slamassociated protein (SAP)-deficient mice on the C57BL/6 background (H-2b) were challenged with a low dose (0.1 LD50 equivalent to 500 EID50) of the mouse-adapted influenza A strain A/PR8 (H1N1) or with a lethal dose (300 LD50) of the A/Philippines/2/82/x-79 (H3N2) strain as in previous studies.24 Heterosubtypic infection was performed on mice that were primed with 0.1 LD50 A/PR8 35 days previously. The attenuated cold-adapted virus ca.A/Alaska (H3N2)25 was originally supplied by S. Epstein (NIH) and was also used in some experiments. All viruses were administered intra-nasally in 50 µL of PBS under light anesthesia. Control mice were administered PBS alone (no virus). All mice were housed in the Lake Nona Vivarium at the


Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Health Sciences campus of UCF and monitored daily for weight loss and general well-being in accordance with IACUC protocols.

Blood was taken from infected and control groups on stated days post-infection (once weekly) by submandibular collection. On the final time point for experiments blood was taken by cardiac puncture. Blood samples were collected into 2.0 mL microcentrifuge tubes. Immediately after obtaining the blood sample, the tubes were placed in an upright position for 1 hour to allow complete blood clotting. The tubes were centrifuged using an Eppendorf Minispin for 5 min at 13400 rpm. The serum was removed to a clean micro cryo vial and used immediately for testing.

Gold nanoparticle test. Citrate-protected gold nanoparticles (AuNP) (15708−9, conc. 5.6 × 109 particles/mL, average diameter 100 nm) was purchased from Ted Pella, Inc. (Redding, CA). Goat anti-mouse IgG (Fc specific) antibody (M4280, lot number 016M4789V) and goat antimouse IgG (Fab specific) antibody (M4155, lot number 055M4809V) was purchased from Sigma-Aldrich (St. Louis, MO). Both antibody products have a concentration of 2.0 mg/mL.

Gold nanoparticle assay was conducted using an automatic NDS1200 DLS instrument from Nano Discovery Inc. (Orlando, FL). This system is equipped with a 633 nm He−Ne laser (0.5 mW) and a 12-sample holder, which allows automatic measurement of 12 samples in 6 min. All size measurements were conducted at an ambient temperature of 25 °C. To perform two-step nanoparticle test, 2 µL of mouse serum was mixed with 40 µL of AuNP. The average particle size of the assay solution was measured using NDS1200 following a 15 and 20 min of incubation at room temperature and the average size value of these two measurements is taken as D1. Then 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 30

µL of goat anti-mouse IgG antibody (2.0 mg/mL) was added to the assay solution. The average particle size of the assay solution was measured again following a 15 and 20 min of incubation and the average size value of these two measurements is taken as D2. The ratio of D2/D1 was calculated as the test score. All blood serum samples were analyzed in duplicates, and error bars presented in each graphs are standard deviations.

ELISA analysis. ELISA for detection of influenza-specific IgG was performed as previously described.26 Briefly, 96 well ELISA plates (Nunc) coated overnight with at 4°C with UVinactivated A/PR8 or A/Alaska virus were washed thoroughly and blocked with PBS containing 2% FBS. Serum samples were serially diluted and incubated overnight at 4°C in the A/PR8coated plates. After thorough washing, HRP-conjugated Ab against mouse IgG was added at 0.2 µg/mL and plates were incubated for 2 hours at room temperature. After thorough washing, the HRP substrate o-phenylenediamine dihydrochloride was added, and the OD of the acid-stopped color reaction measured at 492 nm.

Statistical Analysis. P values as presented in the figures were determined by two-tailed unpaired Student’s t test using GraphPad Prism software. P values < 0.05 were considered as significant difference. The numbers of asterisks indicate significance levels of P values, for example, the symbols of *, **, *** and **** represent P value ≤ 0.05, ≤ 0.01, ≤ 0.001 and ≤ 0.0001, respectively. If there is no significant difference (P > 0.05) between the groups, the results are presented as “ns”, namely not significant.


Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION The kinetics of the murine Ab response to IAV infection is well documented. Following a sublethal primary infection, i.e., when a host is exposed to the virus for the first time, virusspecific IgG appears approximately on day 10 and peaks around day 21.26 The virus is cleared around 14 days post-infection but virus-specific IgG titers remain high indefinitely.26 This is similar to findings in human samples that found the half-life of pathogen-specific IgG to often be extremely long.2 Virus-specific Ab responses have been traditionally monitored by measuring antibody titer using ELISA. Here we also performed ELISA analysis of the collected blood serum samples for comparison with the nanoparticle test. We are reporting the results of four sets of experiments: the first experiment was to examine the immune response following a primary IAV infection; the second experiment was to compare the immune response of a wild-type (WT) mouse model with an immune-compromised SAP KO mouse model in which IgG antibody responses are compromised;26 in the third experiment, we tested the immune response of both A/PR8-primed WT and SAP KO mice following a secondary infection with heterosubtypic IAV (A/Philippines). This heterosubtypic infection models encounter of pandemic IAV in individuals that are successfully vaccinated against a seasonal strain as the A/PR8-primed mice contain neutralizing antibody specific for the priming virus (H1N1) but not against the challenge virus (H3N2). Finally, in a fourth experiment, we infected the WT and SAP KO mice with a cold-adapted attenuated IAV strain. The cold-adapted virus is a vaccine strain that has been shown to prime strong immunity in mice upon challenge with lethal doses of WT virus,27 but because of its attenuation the cold-adapted virus generates much less viral antigen during infection than infection with WT IAV.28



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

In the first set of experiments, 8 wild-type C57BL/6 mice (WT) were challenged with a low dose (0.1 LD50 equivalent to 500 EID50) of A/PR8 (H1N1), and 6 uninfected mice were used as negative control in the study. From both the nanoparticle test and ELISA analysis, no positive response was observed in any uninfected control mice on any given day. The average nanoparticle test score obtained from 6 control mice, 8 blood samples and 16 tests was 1.98, with a standard deviation of 0.32. The test results of one typical negative control mouse are presented in Figure 2A. Among the eight mice in the positive group, seven showed positive immune responses in both the nanoparticle assay and ELISA, and the results for four mice with the strongest responses are shown in Figure 2 (panel B-nanoparticle test results, and panel C-corresponding ELISA results). The test results of the other three positive mice are included in the Supporting Information, Figure 1S. A test is defined as positive if the test score is at least two times of standard deviation above the average score of the negative control group, which is 2.62. Between approximately day 14-21, the nanoparticle test score of the 4 infected mice increased significantly to above 3.0, and some reached to close to 7.0 (Figure 2, panel B). The level of IAV-specific IgG as measured by ELISA also increased accordingly for these 4 mice around the same time (Figure 2, panel C). However, there is a drastic difference between the nanoparticle test and ELISA analysis results: after peaking at around day 21-24, the nanoparticle test score decreased and eventually returned to baseline level around or below 2.5 by day 29-32, while the antibody titer as measured by ELISA remained high and constant well after the infection is cleared, a trend typical of the IgG response induced by IAV and other acute viral infections. Thus, the nanoparticle test appears to give positive test results only during the first few weeks following viral infection.


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this experiment, we prepared a total 8 mice for infection. When performing infection, it was observed that one mouse was not infected properly due to failed anesthesia. However, we kept this mouse in the study as an additional control. This information and the identity of the mouse was not provided to the researchers who conducted the blood analysis. From blood analysis, it was found that this mouse was indeed not infected: both nanoparticle test and ELISA analysis returned complete negative test results from this mouse. The nanoparticle test score of this mouse never exceeded 2.0, and there was no virus-specific antibody production according to ELISA analysis. In summary, there is a 100% correlation between the nanoparticle test and ELISA in the identification of infected mice. In a second set of experiments, 6 wild type WT and 4 slam-associated protein-deficient mice (SAP KO) were infected with a sublethal dose of the same IAV. SAP KO mice demonstrate compromised IgG antibody responses upon IAV priming. While their initial IgG response is similar to that of WT mice, long-lived IgG antibody present at 28 days and later in SAP KO mice is decreased by at least one log as determined by ELISA.26 This is due to compromised follicular CD4 T cell help for the generation of long-lived plasma cells.26,29 We thus hypothesized that serum analysis from the SAP KO mice infected with IAV might also yield different results from wild type mice in the nanoparticle assay. Kinetic analysis of blood samples collected at different time points post A/PR8-infection revealed exactly the same observation as made in the first set of the experiments: the nanoparticle test started to give positive results on day 14 following the infection, peaks at around day 21, and decreases again by day 28. Both WT and SAP KO mice show similar trend (Figure 3A). However, there is also a clear difference between the two mouse models: test scores of the SAP KO group are significantly lower than the WT group (Figure 3B). We also conducted



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

antibody titer analysis using ELISA on day 28 blood samples. The total PR8-specific IgG of the SAP KO group is more than 1 order of magnitude lower than the WT group (Figure 3C), as expected, and serum dilution analysis confirmed the same results (Figure 3D). Again, there is a complete correlation between the nanoparticle test score and the antibody titer as determined by ELISA. This result indicates the strong possibility that the sensitivity of the nanoparticle assay may be directly tied to the amount of, or quality of, anti-viral IgG present in the circulation. In a third set of experiments, the ten mice infected in the second set of experiments were rechallenged with a lethal dose of the IAV strain A/Philippines 35 days post-priming with A/PR8. The immune response following this secondary heterosubtypic challenge is much accelerated versus that generated against a primary IAV infection, with viral clearance within 10 days of infection.27 The enhanced response is due largely to memory CD4 and CD8 T cells that respond to internal viral proteins that are conserved between the priming and challenge virus.27 We have also shown that IAV-primed memory CD4 T cells enhance the kinetics of IgG antibody production following IAV challenge.28 The enhanced secondary immune response coupled with a high heterosubtypic viral challenge dose results in higher levels of viral antigen to be present, combined with higher levels of antibody to be present earlier than seen in the primary response. We thus hypothesized that these conditions would result in an earlier positive test score for the nanoparticle assay than is observed during the primary infection, when positive test scores are only evident beginning at day 14 post-infection. Indeed, from the re-challenged WT mice, nanoparticle testing revealed a clear positive response from day 6 blood samples (Figure 4). Interestingly, a positive nanoparticle test response was not observed from the SAP KO mice on day 6. As the titer of virus-specific IgG antibody is at least an order of magnitude lower in the SAP KO mice as compared to the WT mice, this result supports that positive test results in the


Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanoparticle assay require the simultaneous presence of relatively high levels of both viral antigen and of virus-specific IgG to be present systemically.30 In the final set of experiments we primed WT and SAP KO mice with the cold-adapted vaccine strain A/Alaska. While infection with cold-adapted IAV viruses generate relatively strong neutralizing antibody-dependent immunity in mice, their immunogenicity is at least 10fold less than corresponding WT viruses due to reduced replication in the respiratory tract.31 We thus hypothesized that although virus-specific IgG would be present in mice infected with A/Alaska, the amount of viral antigen would be severely decreased. Indeed, recent experiments showed much reduced antigen-dependent memory T cell formation in mice immunized with A/Alaska versus with WT IAV.32 In mice that were primed with A/Alaska, we observed robust virus-specific IgG production that peaked at day 21 and remained high thereafter (Fig 5A), but the nanoparticle test score did not yield a positive result at any timepoint (Fig 5B). This result supports the hypothesis that the nanoparticle test requires the presence of high levels of viral antigen and anti-viral IgG to generate positive scores, and clearly demonstrates its ability to deliver profoundly distinct results (WT versus attenuated virus infection) in situations where the ELISA results are virtually identical. The above experiments have been repeated multiple times and the results are highly reproducible. The nanoparticle test has excellent analytical reproducibility as demonstrated from the low standard deviation of each data point presented here. Such excellent reproducibility of the nanoparticle test is attributed to the exceptionally strong light scattering property of AuNP as we investigated and discussed previously.17,33 All the experimental data has pointed to one conclusion: the nanoparticle test we reported here appears to give only positive results when



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

there is an active or recent acute viral infection. As the primary immune response against the virus resolves, the nanoparticle test result rapidly returns to negative. The fact that the nanoparticle test only gives positive response when there is an active infection and shortly thereafter is rather intriguing. All the experimental evidence so far suggests that the test requires the co-presence of both virus antigens and virus-elicited antibodies to give positive results. The fourth set of experiment as discussed in prior section strongly supports a model in which the presence of a significant quantity of viral antigen in the system is necessary for a positive nanoparticle test response. The co-requirement of immune active, virus-elicited IgG antibodies for a positive nanoparticle test result is supported by two pieces of evidence: (1) From the analysis of a large number of non-infected control mice in all three sets of experiment as discussed here and in other repeating experiments of which the data are not presented, there was not a single time that the negative control mice gave positive test results. IgG is one of the most abundant proteins in blood, with an average concentration in the 2-5 mg/mL range in mouse blood. However, without an active infection and without the production of immune active IgG, only limited IgG molecules are present in the protein corona, leading to rather low test scores using uninfected mouse serum samples. The increased amount of IgG that is detected from the infected mice serum must be from those immune active IgG antibody produced following viral infection. (2) The results of the nanoparticle test performed on the SAP KO mice versus the WT mice established a positive correlation between the quantity of viral infection-elicited, immune active IgG and the nanoparticle test score. The primary anti-A/PR8 immune response of the SAP KO mice is largely intact, with no differences observed in viral titer between SAP KO and WT mice (not shown). However, the amount of immune active IgG produced by infected SAP KO mice is one order of magnitude lower than the wild-type mice, as


Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


confirmed by ELISA. The lower nanoparticle test scores from the SAP KO mice are thus most likely caused by reduced concentration of immune active IgG specific for the infecting virus. All the experimental observations we have made so far point to a most plausible explanation on the mechanism of the nanoparticle test: viral antigens are first or preferably adsorbed to the AuNP surface, most likely along with other serum proteins and biomolecules, and then antigenspecific IgG Ab become part of the outer layer protein corona by specific binding to the adsorbed antigens as highlighted in Figure 6A. This hypothesis is supported by the analysis of the orientation of mouse IgG present in the protein corona. When conducting nanoparticle analysis of the blood serum samples, we initially used two distinct goat anti-mouse IgG Ab clones: one is specific to the Fab fragment, and another one is specific to the Fc fragment of mouse IgG. Only the Fc specific anti-mouse Ab revealed positive immune response from infected mice, while the anti-mouse IgG (Fab) specific Ab failed to give positive test scores (Figure 6B). The antigenbinding activity of both Abs were certified by the vendor, further confirmed in this study and proved to have similar level of antigen binding activities (Supporting Information). The difference obtained from the two specific anti-mouse IgG Abs used in the nanoparticle test is a clear indication that the Fc fragment of mouse IgG present in the protein corona is oriented toward the outside of the corona, while the Fab fragment is oriented toward the AuNP surface. It should be mentioned here that the viral antigens required to mediate a positive nanoparticle test score could be intact proteins and other biomolecules shed from virus particles, but more likely are processed antigens such as small peptides and other fragments generated during an active immune response. These processed antigens, because of their smaller molecular sizes and exposed hydrophobic moieties, should more readily adsorb to the AuNPs. The formation of such an antigen layer then attracts the adsorption of active anti-antigen Ab through affinity binding.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

Alternatively, the processed viral antigens could have already formed immune complexes with their specific anti-viral antibodies in blood and the adsorption of the whole immune complexes to the AuNP will naturally expose the Fc fragment of the IgG antibody to the AuNP surface, if the virus antigen portion is preferably adsorbed to the AuNP metal core. If the Fc fragment of the IgG antibody in the immune complex is first attached to the AuNP metal core, the IgG would not be detected by the nanoparticle test, because the Fab fragment will be blocked by the bound antigen.

CONCLUSIONS In summary, we report here a novel nanoparticle test that could be of tremendous clinical value for the timely detection and diagnosis of infectious diseases in two major aspects. First, this test addresses the false positive problem of traditional serological test in viral infection diagnosis. While ELISA-based assays detect pathogen-specific antibody produced following virus exposure, a positive result cannot determine whether the antibody is produced from an ongoing, or recent viral infection versus from a past, historical infection. As clearly demonstrated in this study also, after the viral infection is cleared from the mice, the antibody titer remains high and it is common that such specific antibody titer will remain constant in the years to come following the infection. Because the nanoparticle test is capable of detecting antibody only within a brief window occurring during the generation and resolution of an acute infection, the assay is able to identify current or very recent encounter with a pathogen from exposure. This capability can add significant diagnostic power when combined with traditional serology in situations when it is necessary to confirm if a patient is completely cleared of a highly contagious or high health risk viral infection such as the case of Zika infection, so that treatment,


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


quarantining or monitoring is no longer needed. Second, the nanoparticle test does not require one to know the identity of the virus. In the case of a local, epidemic or pandemic outbreak of unknown, new virus, this test can be easily and quickly dispatched to the exposed areas for fast screening of potentially infected populations. Subsequently, the suspected patients and populations can be isolated following a confirmative diagnosis. Because the nanoparticle test comprises a very simple two-step solution mixing process, the test requires only a few microns of blood serum samples, the test can be readily adapted for point-of-care clinical applications.

Supporting Information Method used to evaluate the antigen-binding activity of goat anti-mouse IgG (Fc specific) antibody and goat anti-mouse IgG (Fab specific) antibody; nanoparticle test results of additional positively infected mice and their corresponding ELISA antibody titer analysis.

Acknowledgement This work is supported by Florida Department of Health, Biomedical Research Program (Funding number 7ZK04), the University of Central Florida and the University of Central Florida College of Medicine.

References 1. Storch, G. A. Diagnostic Virology. Clin. Infect. Dis. 2000, 31, 739-751. DOI: 10.1086/314015 2. Slifka, M. K. Immunological Memory to Viral Infection: Commentary. Curr. Opin. Immunol. 2004, 16, 443-450. DOI: 10.1016/j.coi.2004.05.013



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

3. Bogoch, I. I.; Brady, O. J.; Kraemer, M. U. G.; German, M.; Creatore, M. I.; Kulkarni, M. A.; Brownstein, J. S.; Mekaru, S. R.; Hay, S. I.; Groot, E.; Watts, A.; Khan, K. Anticipating the International Spread of Zika Virus from Brazil. Lancet 2016, 387, 335-336. DOI: 10.1016/S0140-6736(16)00080-5 4. Mlakar, J.; Korva, M.; Tul, N.; Popović, M.; Poljšak-Prijatelj, M.; Mraz, J.; Kolenc, M.; Rus, K. R.; Vipotnik, T. V.; Vodušek, V. F.; Vizjak, A.; Pižem, J.; Petrovec, M.; Županc, T. A. Zika Virus Associated with Microcephaly. N. Engl. J. Med. 2016, 374, 951-958. DOI: 10.1056/NEJMoa1600651 5. Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo, Q. A One-Step Homogeneous Immunoassay for Cancer Biomarker Detection Using Gold Nanoparticle Probes Coupled with Dynamic Light Scattering. J. Am. Chem. Soc. 2008, 130, 2780-2782. DOI: 10.1021/ja711298b 6. Dai, Q.; Liu, X.; Coutts, J.; Austin, L.; Huo, Q. A One-Step Highly Sensitive Method for DNA Detection Using Dynamic Light Scattering. J. Am. Chem. Soc. 2008, 130, 8138-8139. DOI: 10.1021/ja801947e 7. Pierre-Pierre, N.; Huo, Q. Dynamic Light Scattering Coupled with Gold Nanoparticle Probes as a Powerful Sensing Technique for Chemical and Biological Target Detection. ACS Symposium Series: Chapter 9, 2015; pp 157-179. Chapter DOI: 10.1021/bk-20151215.ch009 8. Driskell, J. D.; Jones, C. A.; Tompkins, S. M.; Tripp, R. A. One-Step Assay for Detecting Influenza Virus Using Dynamic Light Scattering and Gold Nanoparticles. Analyst 2011, 136, 3083-3090. DOI: 10.1039/c1an15303j


Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9. Wang, L.; Zhu, Y.; Xu, L.; Chen, W.; Kuang, H.; Liu, L.; Agarwal, A.; Xu, C.; Kotov, N. A. Side-by-Side and End-to-End Gold Nanorod Assemblies for Environmental Toxin Sensing. Angew. Chem. Int. Ed. 2010, 49, 5472-5475. DOI: 10.1002/anie.200907357 10. Jans, H.; Liu, X.; Austin, L.; Maes, G.; Huo, Q. Dynamic Light Scattering as a Powerful Tool for Gold Nanoparticle Bioconjugation and Biomolecular Binding Studies. Anal. Chem. 2009, 81, 9425-9432. DOI: 10.1021/ac901822w 11. Jaganathan, S.; Yue, P.; Paladino, D. C.; Bogdanovic, J.; Huo, Q.; Turkson, J. A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells. PLoS One 2011, 6, e19605. DOI: 10.1371/journal.pone.0019605 12. Lai, Y. H.; Koo, S.; Oh, S. W.; Driskell, E. A.; Driskell, J. D. Rapid Screening of AntibodyAntigen Binding Using Dynamic Light Scattering (DLS) and Gold Nanoparticles. Anal. Methods 2015, 7, 7249-7255. DOI: 10.1039/C5AY00674K 13. Bogdanovic, J.; Colon, J.; Baker, C.; Huo, Q. A Label-Free Nanoparticle Aggregation Assay for Protein Complex/Aggregate Detection and Study. Anal. Biochem. 2010, 405, 96-102. DOI: 10.1016/j.ab.2010.06.008 14. Wang, X.; Li, Y.; Quan, D.; Wang, J.; Zhang, Y.; Du, J.; Peng, J.; Fu, Q.; Zhou, Y.; Jia, S.; Wang, Y.; Zhan, L. Detection of Hepatitis B Surface Antigen by Target-Induced Aggregation Monitored by Dynamic Light Scattering. Anal. Biochem. 2012, 428, 119-125. DOI: 10.1016/j.ab.2012.06.011 15. Seow, N.; Tan, Y. N.; Yung, L-Y. L. Gold Nanoparticle–Dynamic Light Scattering Tandem for the Rapid and Quantitative Detection of the let7 MicroRNA Family. Part. Part. Syst. Charact. 2014, 31, 1260-1268. DOI: 10.1002/ppsc.201400158



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

16. Huo, Q.; Cordero, A.; Bogdanovic, J.; Colon, J.; Baker, C. H.; Goodison, S.; Pensky, M. A Facile Nanoparticle Immunoassay for Cancer Biomarker Discovery. J. Nanobiotechnol. 2011, 9, 20-31. DOI: 10.1186/1477-3155-9-20 17. Zheng, T.; Pierre-Pierre, N.; Yan, X.; Huo, Q.; Almodovar, A. J. O.; Valerio, F.; RiveraRamirez, I.; Griffith, E.; Decker, D. A.; Chen, S.; Zhu, N. Gold Nanoparticle-Enabled Blood Test for Early Stage Cancer Detection and Risk Assessment. ACS Appl. Mater. Interfaces 2015, 7, 6819-6827. DOI: 10.1021/acsami.5b00371 18. Zhang, S.; Moustafa, Y.; Huo, Q. Different Interaction Modes of Biomolecules with CitrateCapped Gold Nanoparticles. ACS Appl. Mater. Interfaces 2014, 6, 21184–21192. DOI: 10.1021/am506112u 19. Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 8, 772-781. DOI: 10.1038/nnano.2013.181 20. Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14265-14270. DOI: 10.1073/pnas.0805135105 21. Dobrovolskaia, M. A.; Patri, A. K.; Zheng, J.; Clogston, J. D.; Ayub, N.; Aggarwal, P.; Neun, B. W.; Hall, J. B.; McNeil, S. E. Interaction of Colloidal Gold Nanoparticles with Human Blood: Effects on Particle Size and Analysis of Plasma Protein Binding Profiles. Nanomedicine 2009, 5, 106-117. DOI: 10.1016/j.nano.2008.08.001


Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22. Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS Nano 2010, 4, 365-379. DOI: 10.1021/nn9011187 23. Rahman, M.; Laurent, S.; Tawil, N.; Yahia, L. H.; Mahmoudi, M. Protein-Nanoparticle Interactions; Springer: New York, 2013. DOI: 10.1007/978-3-642-37555-2 24. McKinstry, K. K.; Strutt, T. M.; Buck, A.; Curtis, J. D.; Dibble, J. P.; Huston, G.; Tighe, M.; Hamada, H.; Sell, S.; Dutton, R. W.; Swain, S. L. IL-10 Deficiency Unleashes an InfluenzaSpecific Th17 Response and Enhances Survival against High-Dose Challenge. J. Immunol. 2009, 182, 7353-7363. DOI: 10.4049/jimmunol.0900657 25. Murphy, B. R.; Chanock, R. M.; Clements, M. L.; Anthony, W. C.; Sear, A .J.; Cisneros, L. A.; Rennels, M. B.; Miller, E. H.; Black, R. E.; Levine, M. M.; Betts, R. F.; Douglas, R. G. Jr.; Maassab, H. F.; Cox, N. J.; Kendal, A. P. Evaluation of A/Alaska/6/77 (H3N2) ColdAdapted Recombinant Viruses Derived from A/Ann Arbor/6/60 Cold-Adapted Donor Virus in Adult Seronegative Volunteers. Infect. Immun. 1981, 32, 693-697. DOI: 00199567/81/050693-05$02.00/0 26. Kamperschroer, C.; Dibble, J. P.; Meents, D. L.; Schwartzberg, P. L.; Swain, S. L. SAP Is Required for Th Cell Function and for Immunity to Influenza. J. Immunol. 2006, 177, 53175327. DOI: 10.4049/jimmunol.177.8.5317 27. Powell, T. J.; Strutt, T.; Reome, J.; Hollenbaugh, J. A.; Roberts, A. D.; Woodland, D. L.; Swain, S. L.; Dutton, R. W. Priming with Cold-Adapted Influenza A Does Not Prevent Infection but Elicits Long-Lived Protection against Supralethal Challenge with Heterosubtypic Virus. J. Immunol. 2007, 178, 1030-1038. DOI: 10.4049/jimmunol.178.2.1030



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

28. Strutt, T. M.; McKinstry, K. K.; Kuang, Y.; Bradley, L. M.; Swain, S. L. Memory CD4+ TCell–Mediated Protection Depends on Secondary Effectors That Are Distinct from and Superior to Primary Effectors. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 2551-2560. DOI: 10.1073/pnas.1205894109 29. Kamperschroer, C.; Roberts, D. M.; Zhang, Y.; Weng, N. P.; Swain, S. L. SAP Enables T Cells to Help B Cells by a Mechanism Distinct from Th Cell Programming or CD40 Ligand Regulation. J. Immunol. 2008, 181, 3994-4003. DOI: https://doi.org/10.4049/jimmunol.181.6.3994 30. Lamere, M. W.; Moquin, A.; Lee, F. E.; Misra, R. S.; Blair, P. J.; Haynes, L.; Randall, T. D.; Lund, F. E.; Kaminski, D. A. Regulation of Antinucleoprotein IgG by Systemic Vaccination and Its Effect on Influenza Virus Clearance. J. Virol. 2011, 85, 5027-5035. DOI: 10.1128/JVI.00150-11 31. Tannock, G. A.; Paul, J. A.; Barry, R. D. Relative Immunogenicity of the Cold-Adapted Influenza Virus A/Ann Arbor/6/60 (A/AA/6/60-ca), Recombinants of A/AA/6/60-ca, and Parental Strains with Similar Surface Antigens. Infect. Immun. 1984, 43, 457-462. DOI: 0019-9567/84/020457-06$02.00/0 32. Bautista, B. L.; Devarajan, P.; McKinstry, K. K.; Strutt, T. M.; Vong, A. M.; Jones, M. C.; Kuang, Y.; Mott, D.; Swain, S. L. Short-Lived Antigen Recognition but Not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells To Become Protective Memory. J. Immunol. 2016, 197, 3936-3949. DOI: 10.4049/jimmunol.1600838 33. Zheng, T.; Bott, S.; Huo, Q. Techniques for Accurate Sizing of Gold Nanoparticles Using Dynamic Light Scattering with Particular Application to Chemical and Biological Sensing


Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Based on Aggregate Formation. ACS Appl. Mater. Interfaces 2016, 8, 21585–21594. DOI: 10.1021/acsami.6b06903



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

Figure 1. Illustration of the principle of gold nanoparticle-enabled blood test for detection of active immune response following viral infection. The test comprises a two-step process: in the first step, a small amount of serum sample is mixed with citrate-AuNP solution. During the 1520 incubation time, proteins in the serum, including virus antigens and the specific antibodies, are adsorbed to the AuNP surface. The average particle size (D1) of the AuNP following serum protein adsorption is measured. In the second step, a species-specific antibody, here is a goat anti-mouse IgG (Fc specific) antibody is added to the assay solution. Following another 15-20 min of incubation, the average particle size of the assay solution (D2) is measured again. A test score defined as the ratio D2/D1, is obtained to evaluate the relative amount of mouse IgG present in the AuNP protein corona.

Goat anti-mouse IgG Mouse serum adsorption Au 15-20 min

15-20 min

v

D0~100 nm D1~150-200 nm

Test Score = D2/D1

D2 >> 150-200 nm


Page 25 of 30

Figure 2. Representative nanoparticle test score of an uninfected negative control mouse (A), nanoparticle test scores (panel B) and corresponding ELISA antibody titer analysis (panel C) of four WT mice infected with A/PR8 virus. Blood samples were collected kinetically on different days following viral infection, with the nanoparticle test and ELISA performed on the same samples.

A Test score

9

Negative Control

7 5 3 1 7 11 15 19 23 27 31 Days post infection

5 3

5 3 1

1 4

670LR

1.2

O.D.

3

3 1

689R

1.2

7 11 15 19 23 27 31 Days post infection 1.2

0.6

0.6

0.3

0.3

0.3

0.3

0

0

0

0

4

9 14 19 24 29 34 Days post infection

4


693LR

0.9

0.9

0.6

693LR

5

7 11 15 19 23 27 31 Days post infection

0.9

0.6

5

9 14 19 24 29 34 Days post infection 692N

1.2

0.9

7

689R

1 4


O.D.

C

7

Test score

7

7

692N

Test score

9

670LR Test score

Test score

9

O.D.

B

O.D.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60






Figure 3. (A) Nanoparticle test results of six WT mice and four SAP KO mice upon infection with a sublethal dose of A/PR8 virus. Five uninfected WT mice were used as negative control. Here the test scores are normalized according to the average test score of all negative control samples to compensate slight differences induced by different batches of commercial goat antimouse IgG antibody used in the test. (B) Test score comparison of WT and SAP KO groups on day 14 and day 21 post-infection. (C) Raw antibody titer analysis by 592 nm absorbance and (D) endpoint titer analysis of A/PR8-specific IgG present in the serum of WT mice and SAP KO mice at day 28 as detected by ELISA. A 3.5

3 2.5 2 1.5 1 0.5

Day 7 Day 14 Day 21 Day 28

3 2.5 2 1.5 1 0.5

1.6

3

****

1.2 0.8 0.4

1 0

0.5

Day 14

1.5 1

Negative Control

Day 21

3000000

End point titer

2 1.5

2

D WT SAP KO

**** Absorbance

2.5

2.5

C

B WT SAP KO Negative control


3

0.5

SAP KO

WT

3.5

Normalized test score




3.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

10

100

1000

10000 100000 1000000

Dilution (1:X)

****

300000

30000

3000

WT

SAP KO


Page 27 of 30

Figure 4. Comparison of the nanoparticle test score (normalized score) between primary infection with A/PR8 virus at day 7 and re-infection with A/Phil virus at day 6 for WT mice, SAP KO mice and negative control. The mice used in this study are the same as used for the study as discussed in Figure 3. The re-infection was done at Day 35 following the primary infection. 2.2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2

****

Day 7 upon primary infection Day 6 upon reinfection

1.8 1.6 1.4 1.2

*

ns

SAP KO

Negative Control

1 0.8

WT



Figure 5. (A) Endpoint titer analysis and (B) nanoparticle test results of three WT mice and two SAP KO mice upon infection with the cold-adapted vaccine strain A/Alaska.

A


End point titer

200000 20000 2000 200 20

WT

B Test score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

9.5 8.5 7.5 6.5 5.5 4.5 3.5 2.5 1.5 0.5

SAP KO


ns ns

WT

SAP KO

Negative Control


Page 29 of 30

Figure 6. (A) A possible mechanism underlying the nanoparticle test. In the first step of assay where AuNP is simply mixed with blood serum, viral antigens are first adsorbed to the AuNP, most likely along with other serum proteins, to form an inner corona antigen layer on the nanoparticle surface. Then, virus-elicited active IgG Ab is adsorbed to the AuNP surface through affinity binding with the pre-adsorbed antigens to form an outer corona layer, exposing the Fc portion of the IgG Ab to the outer surface of the nanoparticle. Only the addition of anti-IgG Fc specific antibody can lead to crosslinking of the AuNPs to form nanoparticle clusters. (B) Nanoparticle test results of three positive IAV-primed WT mice using anti-mouse IgG Fc specific Ab versus Fab specific Ab in the second step of the assay. A Antibody layer Antigen layer

AuNP

Anti-mouse IgG (Fc specific)

B 7 Anti-mouse IgG (Fc specific) Anti-mouse IgG (Fab specific)

6

Test score

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5 4 3 2 1

689 R

693 LR

688 L



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 30

For Table of Contents Use Only Active immune response Mouse serum adsorption

Goat antimouse IgG

Au

v

D0 ~ 100 nm D1 ~ 150-200 nm

Nanoparticle Test Score = D2/ D1

D2 >> 150-200 nm


A Rapid Blood Test To Determine the Active Status and Duration of

Recommend Documents