Comparison of Neutralizing Antibody Titers Elicited by mRNA and 1 Adenoviral Vector Vaccine against SARS-CoV-2 Variants 2

23 The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding 24 possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by 25 mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped 26 viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest 27 neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas 28 Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were 29 of low neutralizing titer (IC 50 <50). The data underscore the importance of surveillance for 30 breakthrough infections that result in severe COVID-19 and suggest the benefit of a 31 second immunization following Ad26.COV2.S to increase protection against the variants. 32

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines from two vaccine platforms have been granted U.S. Food and Drug Administration (FDA) Emergency Use Authorization: mRNA-based (Pfizer and Moderna) and adenoviral vector-based (Johnson & Johnson (J&J)), all of which have been shown to be highly effective.The mRNA-based vaccines were 94-95% effective in preventing COVID-19 1 whereas the adenoviral vector-based J&J vaccine had 66.9% efficacy in preventing moderate to severe disease 2 .However, the ongoing emergence of highly transmissible variants with mutations in the spike protein raises concerns regarding possible decreases in vaccine effectiveness due to spike protein antigenic variability.Lambda (C.37) 3 .The increased transmissibility and/or pathogenicity of the variants is due, at least in part, to mutations in the spike protein RBD that increase its affinity for ACE2 on target cells.Mutations in the Beta, Gamma and Delta variant spike RBDs have been shown to cause partial resistance to neutralization by the serum antibodies of vaccinated and convalescent individuals and therapeutic monoclonal antibodies [4][5][6][7][8][9][10][11] .

SARS-CoV
This study compared the neutralization titers of serum antibodies from individuals immunized with three U.S. FDA Emergency use authorization vaccines (BNT162b2, mRNA-1273 and Ad26.COV2.S) against viruses with the VOC and Lambda spike proteins.
The study groups were controlled for age, clinical co-morbidity, history of pre-vaccination infection and sera were collected on similar days post-vaccination.The results demonstrate a high level of cross-neutralization by antibodies elicited by BNT162b2 and mRNA-1273 on the variants but significantly decreased neutralization by those elicited by the single dose Ad26.COV2.S.
Variant pseudotyped lentiviruses.The Delta plus spike contains K417N, L452R and T478K in the RBD (Figure S1A).The Lambda spike protein contains novel L452Q and F490S mutations in the RBD (Figure S1A).We previously described the production of lentiviruses pseudotyped by the Alpha, Beta, Gamma and Delta spike proteins and here report the generation of pseudotypes with the Delta plus and Lambda variant spike proteins and the individual constituent mutations.The variant spike proteins were well expressed, proteolytically processed and incorporated into lentiviral virions at a level similar to that of the parental D614G spike protein in the producer cells and virions (Figure S1B).The measurement of neutralizing antibody titers with such pseudotypes has been shown to yield results consistent with those obtained with the live virus plaque reduction neutralization test 12 .

Reduced sensitivity of virus with variant spikes to neutralization by convalescent
sera and mRNA vaccine-elicited antibodies.Sera from individuals who had been infected prior to the emergence of the variants (collected 32-57 days post symptom onset) neutralized virus with the D614G spike protein with an average IC50 titer of 346 and neutralized the Alpha variant with a similar titer (IC50 of 305).Neutralizing titers for Beta, Delta, Delta plus and Lambda variants were decreased 3.2-4.9-foldrelative to D614G, indicative of a modest resistance to neutralization (Figure 1A, Table S1).The sera of individuals vaccinated with BNT162b2 and mRNA-1273 that were collected 7-days postsecond injection -a peak antibody response timepoint -neutralized virus with the D614G spike with significantly higher titer (1835 and 1594, respectively) relative to the convalescent sera, and the antibodies cross-reacted on the variants with a modest 2.5-4.0-folddecrease in titer (Figure 1A).The resistance of the Beta variant was attributed to the E484K mutation whereas resistance of the Delta variant was attributed to the L452R mutation (Figure S2).The resistance of the lambda variant was attributed to both the L452Q and F490S mutations (Figure S2).
Resistance of viruses with variant spike proteins to neutralization by Ad26.COV2.Selicited antibodies.We next compared the neutralizing titers of antibodies elicited by the BNT162b2 and mRNA-1273 mRNA vaccines with that of the Ad26.COV2.S adenoviral vector-based vaccine.The sera analyzed were collected from individuals at similar timepoints post-final injection, on average (90 days for BNT162b2, 80 days for mRNA-1273 and 82 days for Ad26.COV2.S; Table S2) and from individuals of similar age and with similar clinical co-morbidities (Table S2).None of the participants had a history of COVID-19 pre-or post-vaccination and all were negative for antibodies against the SARS-CoV-2 N protein (Table S2).The results showed that BNT162b2 sera neutralized virus with the D614G and Alpha spikes with an average titer of 695 and 626.Compared to the D614G, the neutralizing titer against Beta was decreased 6.1-fold and Delta plus was decreased 2.7-fold.Results for the mRNA-1273 vaccine were similar with a 3.3-fold decrease in neutralizing titer for Delta plus and 4.6-fold for Beta.Ad26.COV2.S sera neutralized D614G and Alpha variants with average IC50 titers of 221 and 232, respectively, and neutralized the variants with titers that were decreased by 5.4-fold for Delta plus to 6.7-fold for the Beta variant as compared to D614G (Figure 1B).
Presentation of the data grouped by variant shows the decreased neutralizing titers against the variants by sera of the Ad26.COV2.S-vaccinated individuals (Figure 1C).The L452R/Q mutation of the Delta plus and Lambda spike proteins increases infectivity and affinity for ACE2.Measurement of the infectivity of the pseudotyped viruses, normalized for particle number, showed that the Lambda variant spike protein increased viral infectivity by 2-fold (Figure 2A), an increase equivalent to that of the Delta and Delta plus variants.The increase was due to the L452Q mutation and was similar to that of the L452R found in the Delta and Delta plus variants.The other mutations (D246-252, G75V-T76I, F490S and T859N) had no significant effect on infectivity (Figure 2A).
Measurement of the relative affinity of the variant spike proteins for ACE2 using sACE2 neutralization assay showed that variant spikes had a 3-fold increase in sACE2 binding (Figure 2B).This increase was confirmed in a virion:ACE2 binding assay (Figure 2C).
The increase was caused by the L452R and L452Q mutation and were similar to the increase caused by the N501Y mutation 13,14 .
Neutralization by REGN10933 and REGN10987.Analysis of REGN10933 and REGN10987 monoclonal antibodies that constitute the REGN-COV2 therapy showed that REGN10933 had decreased activity against the Beta variant spike which resulted in a 127-fold decrease in neutralizing titer.REGN10933 also had decreased activity against the Delta plus variant which resulted in a 92.7-fold decrease in neutralizing titer.The resistance to REGN10933 was attributed to K417N and E484K (Figure S3).REGN10933 neutralized virus with the Delta variant spike with a 12-fold decrease in titer which had only a minor effect on the activity of the cocktail.REGN10987 showed a minor reduction in neutralizing titer of virus with the Beta, Delta, Delta plus and Lambda variant spikes but

Discussion
Several reports have shown partial resistance of SARS-CoV-2 VOCs to vaccine-elicited antibodies [4][5][6][7][8][9][10][11] .The data shown here extend those findings to the Delta plus and Lambda variants.Delta plus and Lambda, VOIs, both displayed a degree of resistance to mRNA vaccine-elicited antibodies similar to that of the Beta and Delta variants.In sera collected ~3 months post-second immunization, BNT162b2 and mRNA-1273 mRNA vaccineelicited antibodies neutralized the variants with a modest 3-fold average decrease in titer resulting in an average IC50 of about 1:600, a titer that is greater than that of convalescent sera and likely, in combination with post-vaccination T-and B-cell memory responses, to provide durable protection.Ad26.COV2.S vaccination-elicited neutralizing antibodies showed a more pronounced decrease in neutralizing titer against the variants, raising the potential for decreased protection against the VOCs and the Lambda variant.Vaccination with Ad26.COV2.S resulted in IC50 titers against Beta, Delta, Delta plus and Lambda variants that decreased 5-7-fold, resulting in mean neutralizing antibody titers of 33, 30, 41, and 36 against viruses with the Beta, Delta, Delta plus and Lambda variant spikes, respectively, which according to mathematical modeling, could result in decreased protection against infection 15 .Modeling predicts that 50% protection from infection is provided by a titer that is 20% that of the mean convalescent titer.In this study, given a mean convalescent titer of 346 (Table S1), 50% protection would correspond to an IC50 of 69.The titer required to protect against severe disease was shown to be 3% that of the mean titer of convalescent sera which in this study corresponds to a titer of 10.In a published report of phase 3 trial data, a single dose of Ad26.COV2.S, 28 days post administration, provided 64.0% protection against moderate to severe disease and 81.7% against severe-critical COVID-19 in a country where 95% of circulating SARS-CoV-2 was the Beta variant 2 .The authors considered possible roles for non-neutralizing antibody Fcmediated effector functions and the role of the T cell response in maintaining protection against the partially neutralizing antibody-resistant Beta variant.
The data reported here differ somewhat from those reported by Barouch et al. and    Jongeneelen et al. who found that Ad26.COV2.S-elicited antibody titers were mostly maintained against the variants 16,17 .In addition, Alter et al. reported a 5-fold decrease in neutralizing antibody titer against Beta and 3.3-fold decrease against the Gamma variant by the sera from Ad26.COV2.S vaccination 18 which were less pronounced than those reported here.While the studies used similar assays to measure antibody neutralization and analyzed sera collected at a similar time-point post-immunization, it is possible that differences in the study populations accounted for the experimental differences.
Several recent studies have shown that boosting a single immunization of the ChAdOx1nCoV-19 adenoviral vector vaccine with BNT162b2 resulted in high neutralizing titer against the VOCs [19][20][21] .It is likely that neutralizing antibody titers against the VOCs elicited by the single shot Ad26.COV2.S could similarly be improved by boosting with a second immunization or by a heterologous boost with one of the mRNA vaccines.While a single dose vaccination has advantages, the benefit provided by a second immunization may be well worth the inconvenience.
The data presented here emphasize the importance of surveillance for breakthrough infections with the increased prevalence of highly transmissible variants.If an increase in breakthrough infections accompanied by severe COVID-19 is found following adenovirus vector or mRNA vaccination, this would provide a rationale for public health policy-makers and manufacturers to consider booster immunizations that would increase protection against the VOCs and Lambda variant.As such a need is not currently evident, the public health apparatus should focus on primary immunization in the U.S. and globally.

Clinical Samples
Convalescent sera were collected 32-57 days post-symptom onset.For the early timepoint, BNT162b2 and Moderna-vaccinated sera were collected on day 28 and 35, respectively, 7 days post-second immunization.For the later time-point, BNT162b2vaccinated sera were on average collected 90 days post-second immunization and mRNA-1273-vaccinated sera were collected on average 80 days post-second immunization.Ad26.COV2.S-vaccinated sera were collected, on average, 82 days postimmunization (Table S2).Blood was drawn at the NYU Vaccine Center with written consent under IRB approved protocols (IRB 18-02035 and IRB 18-02037).REGN10933 and REGN10987 were generated as previously described 22 .
The amount of virus bound was quantified by immunoblot analysis of bound p24.

Figure 1 .
Figure 1.Comparison of neutralization titers of variant spike protein pseudotyped

Figure 2 .
Figure 2. Neutralization of variant spike protein pseudotyped viruses by

(
C) Neutralizing titers of serum samples from mRNA-1273 vaccinated donors (n=6).The serum was collected at early time point (7 days after second immunization).The neutralization IC50 from individual donors is shown.Significance is based on the two-sided t-test.Supplemental Figure S3.Neutralization titers of spike protein pseudotyped viruses (single point mutations) by monoclonal antibodies.Neutralization of variant spike protein variants (single point mutations) by REGN10933 and REGN10987 monoclonal antibodies.The IC50 of REGN10933, REGN10987 and the cocktail is shown in the Figure1