key: cord-0941571-p657h6q2
authors: Narowski, Tara M.; Raphel, Kristin; Adams, Lily E.; Huang, Jenny; Vielot, Nadja A.; Jadi, Ramesh; de Silva, Aravinda M.; Baric, Ralph S.; Lafleur, John E.; Premkumar, Lakshmanane
title: SARS-CoV-2 mRNA Vaccine Induces Robust Specific and Cross-reactive IgG and Unequal Strain-specific Neutralizing Antibodies in Naïve and Previously Infected Recipients
date: 2021-06-19
journal: bioRxiv
DOI: 10.1101/2021.06.19.449100
sha: 0f61f760cc55d7e7fee7215a7143d8380c6fac0c
doc_id: 941571
cord_uid: p657h6q2

With the advance of SARS-CoV-2 vaccines, the outlook for overcoming the global COVID-19 pandemic has improved. However, understanding of immunity and protection offered by the SARS-CoV-2 vaccines against circulating variants of concern (VOC) is rapidly evolving. We investigated the mRNA vaccine-induced antibody responses against the referent WIV04 (Wuhan) strain, circulating variants, and human endemic coronaviruses in 168 naïve and previously infected people at three-time points. Samples were collected prior to vaccination, after the first and after the second doses of one of the two available mRNA-based vaccines. After full vaccination, both naïve and previously infected participants developed comparable robust SARS-CoV-2 specific spike IgG levels, modest IgM and IgA binding antibodies, and varying degrees of HCoV cross-reactive antibodies. However, the strength and frequency of neutralizing antibodies produced in naïve people were significantly lower than in the previously infected group. We also found that 1/3rd of previously infected people had undetectable neutralizing antibodies after the first vaccine dose; 40% of this group developed neutralizing antibodies after the second dose. In all subjects neutralizing antibodies produced against the B.1.351 and P.1 variants were weaker than those produced against the reference and B.1.1.7 strains. Our findings provide support for future booster vaccinations modified to be active against the circulating variants.

The SARS-CoV-2 virus causes a spectrum of disease from asymptomatic to severe forms with high mortality. The nucleocapsid protein encapsulating viral RNA and the surface exposed spike protein are the primary targets of human antibodies. The spike protein of SARS-CoV-2 mediates virus attachment and entry into host cells. It comprises a highly variable S1 segment, which harbors the Nterminal domain (NTD) and the receptor-binding domain (RBD), and a more conserved S2 segment which includes the fusion peptide and heptad repeats required for virus fusion to host cells. Neutralizing antibody response is at present the best correlate of protection 1 . However, the adaptive immune response to SARS-CoV-2 infection is variable [2] [3] [4] . RBD accounts for ~90% of the neutralizing activity in SARS-CoV-2 immune sera 5, 6 . RBD-specific antibodies target distinct antigenic sites and exert neutralizing activity principally by interfering with spike protein interactions with its cognate receptor, angiotensin-converting enzyme 2 (ACE2). A subset of the NTD-specific antibody also neutralizes SARS-CoV-2 by targeting a supersite, possibly preventing proteolytic activation, membrane fusion, or spike protein interactions with an auxiliary receptor 7 . The spike protein has been targeted in most SARS-CoV-2 vaccines under development and in those approved and currently being administered worldwide.

The recent effort to achieve widespread vaccination against SARS-CoV-2 has left in its wake a host of questions about whether the vaccine can protect against SARS-CoV-2 infection and whether the vaccine can boost immunity in previously infected people [8] [9] [10] . Infection with four human endemic coronaviruses (HCoVs; OC43, HKU-1, NL63, and 229E) are quite common, and most adults have antibodies to these viruses 8, 9 . Induction of cross-reactive HCoV antibodies has been reported in SARS-CoV-2 infection, and after vaccination 11 . While their role in protection or immunopathogenesis remains unclear 12, 13 , levels of HCoV cross-reactive antibodies correlate with disease severity 14 . The emergence of new, increasingly infectious and virulent SARS-CoV-2 variants is causing significant concern in global human health. The US SARS-CoV-2 Interagency Group has classified the variants currently circulating in the United States, including London (B.1.1.7), Brazil (P.1.), South African (B.1.351), and California strains (B.1.429 and B.1.427), as variants of concern (VOC); these more transmissible, virulent strains are becoming dominant within populations rapidly. VOCs have accumulated key mutations, particularly in the spike protein within the NTD and the RBD, and significant concerns are developing around the efficacy of currently available treatments and vaccines. Understanding factors that underly the level of defense provided by SARS-CoV-2 vaccines against the reference WIV04 strain and the circulating variants of concern is an urgent priority 15 .

We previously reported that seroprevalence among a cohort of uniformly exposed emergency department health care providers was about 5% after the first peak of the infection in Washington, DC, in the late Spring of 2020 16 . PCR confirmed documented SARS-CoV-2 infection increased to about 11% in this cohort of 237 participants by the end of 2020, an incidence of infection in line with health care providers generally in North America 17 . Here we investigate the longitudinal antibody response to the reference Wuhan strain (WIV04), the emerging variants of concerns, and the four HCoVs at threetime points: the Spring of 2020 after the pandemic's first peak; January of 2021 in the period immediately following the roll-out of the mRNA vaccines; and after the majority of the cohort had been fully vaccinated in early March 2021. By combining PCR and serology test results, we first determined that the previous exposure to SARS-CoV-2 irrespective of the symptoms within this cohort is about 14% before vaccination. We then evaluated SARS-CoV-2 mRNA vaccine-induced antibody isotypes, the magnitude of spike-specific and cross-reactive antibodies, and neutralizing antibodies against the reference WIV04 strain and the circulating VOCs.

and March 2021, we enrolled 237 healthcare workers in a large tertiary academic medical center (George Washington University, United States), to estimate baseline serostatus and study antibody response after doses 1 and 2 of an mRNA vaccine among previously infected and naïve individuals (Supplementary Figure 1 and table 1) . Overall, participants were young (median age=30 years) and healthy (82.1% reported no chronic medical conditions). Of 237 healthcare workers, 161 participants received the Pfizer-BioNTech mRNA vaccine (BNT162b2), and 7 participants received then Moderna mRNA-1273 vaccine. Both of these vaccines use mRNA to induce the expression of stabilized fulllength SARS-CoV-2 spike protein 18 . We collected a total of 424 longitudinal samples from the 237 health care workers. This was done pre-vaccination (n=136 collected 6 months before vaccination), after dose 1 (n=149 collected between 6 -28 days of vaccination [median 21 days]), and after dose 2 were previously infected, and 148 were naïve. Analysis of self-reported symptoms following vaccination indicated that naïve individuals tended to experience fewer symptoms following both vaccine doses than previously infected individuals ( Supplementary Figures 2-3 ).

In symptomatic SARS-CoV-2 infections, IgG, IgM, and IgA antibodies are typically developed after 9 days post symptom onset 2 . To understand the antibody response following the doses 1 and 2 mRNA vaccines, we measured the SARS-CoV-2 antibody isotypes against full spike and RBD antigens ( Figure 1 ).

Among the naïve, antibody levels were low up to 7 days, and IgG antibodies to full spike and RBD antigens seroconverted in >95% of the participants by day 8 (Figures 1A and D) . The predominant response was for the IgG antibodies in both naïve and previously infected participants after doses 1 and 2. Among naïve individuals, IgM and IgA antibodies were higher after the first dose than the second dose ( Figure 1G and H). In previously infected individuals, IgM levels were generally lower than the naïve individuals. However, IgA levels in the previously infected individuals were comparable to the naïve individuals after dose 1, and was unchanged after dose 2 ( Figure 1H ).

Having observed that the IgG antibodies are dominant, we compared the magnitude of IgG response after doses 1 and 2 in titration ELISA assays against full spike, RBD, and NTD antigens ( Figure 2 ). The spike IgG antibody levels, measured by the area under the curve in titration experiments, was robustly boosted among the previously infected participants following dose 1, but this response varied in some individuals (Figure 2A -C). The variation of the spike IgG antibodies after the first dose was higher in naïve than the previously infected individuals. After dose 2, IgG response to spike antigens was highly focused, and the antibody levels were comparable between naïve and the previously infected individuals. The magnitude of RBD antibodies correlated well to NTD and the full spike antibodies in both groups ( Figure 2E and F).

We and others have previously shown that the levels of RBD binding antibodies correlated to the SARS-CoV-2 neutralizing antibody titers [19] [20] [21] [22] . To understand the relationship between neutralizing antibodies and spike binding antibodies after mRNA vaccination, we measured live virus SARS-Cov-2 neutralizing titers in 37 paired samples comprising 15 previously infected and 22 naïve individuals after vaccine doses 1 and 2 ( Figure 3 ). Among previously infected and naïve vaccine recipients, we observed a robust correlation between SARS-CoV-2 neutralizing antibodies and the levels of the spike RBD and NTD IgG binding antibodies and a modest correlation with full-spike binding IgG antibodies ( Figure 3A and B). We also noticed a moderate-to-high correlation between the neutralizing antibody levels and RBD and full-spike serum IgA antibodies among previously infected vaccine recipients. Strikingly, about 80% (19/22) of the naïve did not develop detectable levels of neutralizing antibodies after dose 1, and about 50% (9/19) of those naive vaccine recipients neutralizing antibodies was not detectable even after dose 2 ( Figure 3B ). In contrast, 66% (10/15) of previously infected individuals developed neutralizing antibodies after dose 1, and after dose 2, 80% (12/15) of the previously infected had neutralizing antibodies ( Figure 3A ). Even though both naive and previously infected individuals developed similar levels of spike binding antibodies after dose 2, the mean and median neutralizing antibody levels among the naive recipients were at least ten and four folds weaker than the previously infected vaccine recipients (Cf. Figure 3A and 3B). Overall, the relationship between spike binding and SARS-CoV-2 neutralizing antibodies typically improved between doses 1 and 2, indicated by the increase in correlation and decreased interquartile range (IQR), and followed nonmonotonic relationships, which we divided into three groups. In group 1, neutralizing antibodies were undetectable after dose 1 and remained undetectable or became detectable after dose 2. In group 2, neutralizing antibody response declined between doses 1 and 2, whereas neutralizing antibody response in group 3 improved between doses 1 and 2.

Neutralizing antibodies developed against WIV04 strain are weaker against other circulating variants. To understand the vaccine effectiveness against the circulating variants (B.1.1.7, B.1.351, and P.1), we analyzed and compared the neutralizing activity for the reference WIV04 strain and the variants after doses 1 and 2 among naive and previously infected individuals in a multiplex surrogate neutralization assay ( Figure 4A and B). The multiplex surrogate neutralization assay simultaneously measured antibodies that can block the interaction between RBD and ACE2 in a panel of spike antigens from the reference WIV04 strain and the three most concerning novel viral variants B.1.1.7, P.1, and B.1.351. The percentage of the ACE2 blocking antibodies in the surrogate neutralization assay with 100X diluted sera robustly correlated to the neutralizing antibody titers obtained from the BSL-3 SARS-CoV-2 neutralization assay ( Figure 4C ). In general, previously infected vaccine recipients displayed higher spike-ACE2 blocking activity compared to the naïve vaccine recipients. The magnitude of RBD binding antibodies correlated with the spike-ACE2 blocking activity against the reference strain and followed a non-monotonic relationship across the three groups as observed for the live virus neutralization assay ( Figure 3 ). The spike-ACE2 blocking activity of B.1.1.7 strains in naïve and preexposed individuals was slightly lower but closely tracked the ACE2 blocking activity of WIV04 ( Figure   4D and E). In comparison, the naïve and previously infected vaccine recipients displayed significantly reduced ACE2 blocking activity against B.1.351 and P.1 strains. Overall, people with high RBD binding antibodies also developed better ACE2 blocking activity and vice versa ( Figure 4D and E).

infected than in naïve individuals. All participants in our cohort have been previously exposed to more than one human endemic CoVs. The development of cross-reactive antibodies to human endemic CoVs was reported previously in hospitalized patients with severe SARS-CoV-2 symptoms 23 . We, therefore, measured longitudinal antibody levels against the full spike antigens from the reference SARS-CoV-2 and the four-human endemic CoVs using a titration ELISA with the samples collected at pre-vaccination and after doses 1 and 2 from previously infected and naïve individuals ( Figure 5 ). While most vaccine recipients developed antibodies to SARS-CoV-2 as expected, we observed that some previously infected and naïve individuals developed strong cross-reactive antibodies to HCoV spike antigens. The cross-reactive antibody levels were more robust after dose 1 against the -HCoVs (OC43 and HKU-1, Figure 5B -C, G-H and K) than -HCoVs (NL63 and 229E, Figure 5D -E, I-J and K). Similarly, the cross-reactive antibody levels against HCoVs were more pronounced in previously infected individuals than the naïve individuals, marked by a sharper rise after dose 1 followed by a noticeable decline between the first and second dose ( Figure 5) . Notably, the levels of cross-reactive antibodies induced after vaccination was highly correlated among HCoVs, indicating that the crossreactive antibodies are most likely targeting the conserved S2 segment of the spike protein ( Figure 5L ).

Our study on binding and neutralizing antibody response after two doses of one of the two available mRNA vaccines in 168 naïve and previously infected individuals provides novel insights into vaccine-induced immunity and protection against reinfection by both the reference strain and the circulating variants of concern. Here we report on a lower-than-expected frequency of neutralizing antibodies among fully vaccinated naïve recipients and also provide evidence that some previously infected people may require more than one mRNA vaccine dose to strengthen the immune response against SARS-CoV-2.

As has also been reported in a few independent studies 24,25 , we observed comparable and robust IgG responses to spike RBD and NTD in both naïve and previously infected people after full immunization with an mRNA vaccine 24, 25 . Previously, we and others have shown that levels of RBD antibodies correlated well to the magnitude of the neutralizing antibodies in symptomatic SARS-CoV-2 infections [19] [20] [21] [22] 26 . Among convalescent plasma donors, who recovered after symptomatic infections, 80% had detectable levels of live virus-neutralizing antibodies 27 ; of those with binding anti-RBD IgG over 1:160 titers, 95% displayed live virus-neutralization. By contrast, in our cohort, even though binding anti-RBD titers were over these thresholds after full immunization, about 2/5 th of the naïve and 1/5 th of the previously infected vaccine recipients did not develop detectable levels of neutralizing antibodies as evaluated in an authentic live-virus neutralization assay.

Threshold levels of neutralizing antibodies for SARS-CoV-2 immunity have yet to be established, and even relatively low levels of neutralizing antibodies have been associated with protection in nonhuman primate models 28 . Nonetheless, the lack of detectable levels of neutralizing antibodies in 40% of naïve participants is concerning and differs from the previous reports 25 . That being said, and while data is scanty, clinical evidence does not yet exist that vaccination of the previously infected confers more immunity to reinfection than does vaccination of naïve recipients to primary infection 29 .

Moreover, while elevated levels of neutralizing antibodies are considered highly protective 30 , some reports have questioned this relationship 31 , and suggested that other biomarkers may better predict SARS-CoV-2 immunity [32] [33] [34] [35] [36] .

Along with neutralizing IgG, the production of anti-spike IgA in the airways has been proposed to prevent SARS-CoV-2-infection 37, 38 . Mucosal immunity plays a vital role in viral respiratory infections;

IgA dominates early immune response with development of mucosally oriented plasmablasts in natural SARS-CoV-2 infection 39 . Neutralizing mucosal IgA has been associated with milder SARS-COV-2, while circulating neutralizing IgG with more severe disease 34 . The importance of mucosal protection in SARS-COV-2 has led some to advocate for nasally administered vaccines that induce more robust mucosal IgA responses 40 . In our study parenterally delivered mRNA vaccines induce high levels of anti-Spike IgG-similar to what is seen in severe disease. IgA production, by contrast, is induced in more modest quantities, comparable to what is seen in mild disease 11 . Induction of strong IgG and, low IgA levels in naïve patients after receiving one of the two mRNA vaccines has also been recently reported in other independent studies 42 . Serum IgA levels are not a direct measure of secretory IgA levels, since systemic IgA are predominantly made of monomeric IgA1 subclass, and mucosal IgA is a polymeric IgA2 subclass 43 . However, induction of mucosal IgA after parenteral mRNA vaccination has been reported 41 . Our data shows that after full vaccination, spike IgA levels in serum correlated better to the levels of neutralizing antibodies in previously infected vaccine recipients than among naïve recipients, a phenomenon that may be related to a more mature immune response in the former group and recall of memory B cells from natural infection.

Multiple reports have surfaced recently that previously infected individuals may not need or benefit from a second dose of an mRNA vaccine 24, [44] [45] [46] [47] [48] . Our data, by contrast, shows that 1/3 rd of previously infected individuals did not produce detectable levels of neutralizing antibodies after the first dose ( Figure 3A , Group 1); after the second dose, however, 40% of these non-responders produced neutralizing antibodies. A lack of increased spike binding antibody level after the second dose in those previously infected with SARS-CoV-2 has also been cited as evidence that the second dose is unnecessary 24, 49, 50 . Our data also shows a stable or declining antibody levels after the second dose in 2/3 rd of previously infected individuals ( Figure 3A , group 2 or 3), a result that has also been noted by other 49, 51, 52 . Binding antibody levels after the first dose in the previously infected tend to increase by orders of magnitude; what our data shows is that in this group while after the second dose of vaccine higher antibody levels tend to decline, lower levels tend to increase, suggesting focusing of the immune response. This observation is strengthened by the fact that the proportion of previously infected subjects with neutralizing antibodies increased even while mean levels of binding antibodies stayed level or decreased, likely an effect of memory B cells, and associated plasma B cells in the previously infected producing more targeted and affinity-matured antibodies 17, 53 .

Compared to naïve recipients, which experience significant increases in the production of variant-neutralizing antibodies after both doses of vaccine, we observed a trend toward decreasing neutralization against the circulating SARS-CoV-2 variants in the previously infected after the second dose. This may be related to immune dominance of 'originally imprinted' antigens that can then weaken the response to subsequently encountered related antigens, as could be the case with the SARS-CoV-2 variants 54, 55 . Others have argued that 'affinity maturation' can over time produce 'broadly neutralizing antibodies' (BNABs) with the ability to provide sterilizing immunity based on high affinity for conserved epitopes among variant pathogens 53, 56 . 'Persistent' exposure to pathogen antigens is thought to underlie the process of affinity maturation and the development of BNABs. While optimized 53 timing of SARS-CoV-2 spike antigen exposure through booster vaccinations with the currently approved mRNA vaccines may be a route to increase resistance to SARS-CoV-2 variants, our data doesn't suggest this. There are reports 58 of the development of adequate neutralizing antibodies against SARS-CoV-2 variants following administration of the existing mRNA vaccines to previously infected subjects.

However, our data suggests that as the immune response to SARS-CoV-2 spike antigen matures in vaccinated subjects, susceptibility to the variants will remain significant, raising the question if variantfocused re-vaccination with modified vaccines will become necessary? There are concerns 55, 59 regarding potential problems with the effectiveness of such modified vaccines; however, there are not at present, strong reasons to think that the current vaccine regimen, or repeated doses of the existing vaccines, are likely to induce BNABs against SARS-CoV-2 variants. By contrast, a potent crossreactive antibody 60 derived from a SARS-CoV-2-variant epitope has recently been reported, suggesting that modified vaccines produced in this fashion may prove more broadly effective. Widely administered booster immunizations against reference/variants will expose a previously vaccinated and sensitized population to risk of vaccine reaction with attendant risks and discomforts 61 

Our data shows a significant lack of neutralizing antibodies in naïve subjects after full vaccination, and a more limited but concerning lack of neutralizing antibodies in some previously infected individuals. In contrast to what has previously been reported, we find that previously infected individuals may benefit from two doses of the currently available mRNA vaccines. Our data does not show improving neutralizing antibodies against circulating variants with repeated vaccine doses in the previously infected, suggesting that booster vaccinations with vaccines modified for the variants may be called for in the future. We also find that nucleocapsid IgG wanes over time and is therefore limited as an assay for previous infection with SARS-CoV-2.

Study limitations include the inclusion of subjects receiving only mRNA vaccines. Our cohort's previously exposed study subjects were unlikely to have been infected with one of the variants. Study participants included primarily young, healthy adults; chronically ill, immunocompromised, and the very young and very old are not represented in our study. Our data represent interim values that are likely to evolve with time in terms of response to vaccination.

In this study, a total of 424 venous blood samples were collected from 237 unique ED HCP participants at George Washington University Hospital (GWUH) during the timeframe of June 2020 to March 2021.

ED HCP participants were defined as any GWUH ED healthcare providers that came into close contact Additionally, in order to reach those not on the listserv, notifications were sent out through GWUH's ED nurse/technician scheduling system and fliers were placed in break rooms with QR codes connecting to patient sign-up forms. Participation days and times overlapped nurse and technician shift changes in order to encourage on-going and off-going staff to participate. All ED HCP personnel who chose to participate in this study provided written informed consent. All personnel who consented to participate were included in the study.

All participants were asked to complete a questionnaire about demographics (age, gender, race/ethnicity, home city), ED HCP occupation, non-ED HCP affiliations (e.g. also work in the ICU), past medical history (PMH), current medications, smoking history, history of known positive COVID-19 status, recent/intercurrent viral syndrome symptoms (fever, fatigue, dry cough, anorexia, body aches, dyspnea, sputum, sore throat, diarrhea, nausea, dizziness, headache, vomiting, and abdominal pain), relative number of COVID-19 exposures at work and outside of work, and personal protective equipment (PPE) wearing habits (time spent wearing and frequency of changing surgical masks, N95 masks, and Powered Air Purifying Respirators. Additionally, participants in the second and third rounds of testing were asked questions regarding COVID-19 vaccine status, type of COVID-19 vaccine received, dates of first and second vaccine doses, and recent/intercurrent viral syndrome symptoms after obtaining COVID-19 vaccinations.

Sample Collection. Venous blood samples were collected from each participant during each round of testing. 10 ML of blood was drawn into an SST tube, and refrigerated overnight to allow for serum separation. Subsequently the serum was drawn off into a 2 ML Eppendorf tube and stored at -80 deg.

C. for subsequent use.

Protein expression and purification. The expression and production of halo-tagged SARS-CoV-2 and the four human endemic CoV RBD antigens from mammalian cells were previously described 22 .

The halo-tagged SARS-CoV-2 NTD antigen (16-305 amino acids, Accession: P0DTC2.1) was designed and expressed in mammalian Expi293 cells as described for RBD antigens. RBD and NTD antigens were site-specifically biotinylated using Halotag PEG biotin ligand (Promega), following the manufacturer's protocol. For producing SARS-CoV-2 spike protein trimer, a codon-optimized synthetic gene was synthesized to encode for a prefusion-stabilized SARS-CoV-2 spike protein (16-1208 amino acids, Accession: P0DTC2.1) with an N-terminal human serum albumin secretion signal peptide and C-terminal T4 foldon trimerization domain, TEV protease cleavage site, His8 tag, and the twin-strep tag.

The prefusion-stabilized SARS-CoV-2 construct contains two consecutive proline substitutions in the S2 subunit as described before (PMID: 32075877, PMID: 32155444). The synthetic gene was cloned between KpnI and XhoI sites of the mammalian expression plasmid pαH. SARS-CoV-2 spike protein was expressed as described for RBD and NTD antigens in Expi293 cells and purified from mammalian cell culture supernatant using Strep-Tactin immobilized affinity resin (IBA Lifesciences). The bacterial expression construct for full-length SARS-CoV-2 nucleocapsid was a gift from Nicolas Fawzi (Addgene plasmid # 157867 ; http://n2t.net/addgene:157867 ; RRID:Addgene_157867) 69 . The MBP fused nucleocapsid protein was expressed and purified from BL21(DE3)PlysS as described before 70 . The 

Characteristics of the sample were summarized using simple descriptive statistics. To describe the magnitude and spread of full spike and RBD binding IgG, IgM, and IgA titers, we generated jittered dot plots stratified by antibody isotype, time of sampling (post dose 1 and post dose 2), and prior exposure to SARS-CoV-2. Line graphs stratified by time of sampling were used to describe trends in change in antibody levels between doses. The strength of the correlations between spike RBD and spike NTD titers, between spike RBD and full Spike titers, between spike binding antibody titers and SARS-CoV-

blocking Ab percent, were evaluated using a two-tailed Spearman's rank correlation in Prism 9.

Neutralizing antibody responses were grouped into Group 1 (undetectable after dose 1 and remained undetectable or became detectable after dose 2), Group 2 (declined between doses 1 and 2), and 

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This study was supported by grants from the National Cancer Institute (U54CA260543 to A.M.de, R.S.B, and L.P.) and National Institutes of Health, Allergy and Infectious Diseases (U01AI151788 to A.M.de and L.P). We would like to thank the contributions of HCP donors and the staff at GWU for this work. We thank Dr. Liu and the members of the Liu Lab at George Washington University's Milken Institute School of Public Health. We would like to acknowledge Meso Scale Discovery for donating plates and reagents to perform the surrogate neutralization assay to this study. We would like to thank the Adams School of Dentistry DELTA Translational ReCharge Center and its Co-directors, Drs.Shannon Wallet and Robert Maile for the infrastructure support to this study. We thank Mr. John Forsberg at the UNC protein expression core facility and Mr. Salman Khan for assisting with protein expression.

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