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Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOCs) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOCs. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses elicited by various vaccine platforms against VOCs, compared with non-human primates or humans, suggesting caution should be exercised when interpreting data obtained with this animal model.


In brief
Understanding vaccine-elicited responses against SARS-CoV-2 variants of concern in real time is key to vaccine design. Walls et al. report that mice vaccination does not recapitulate the breadth and potency of neutralizing responses seen in non-human primates and humans.

INTRODUCTION
Vaccine candidates are typically evaluated in small mammals (e.g., mice) and non-human primates (NHPs) prior to cGMP manufacturing and human clinical trials. For SARS-CoV-2, serum neutralizing antibody titers represent the current best correlate of protection in NHP challenge studies (Arunachalam et al., 2021;Corbett et al., 2021) and in humans (Gilbert et al., 2021). Serum neutralizing antibody titers are also used as metrics in human clinical trials to benchmark new vaccine candidates (e.g., NCT05007951 comparing GBP510 to AZD1222) and the administration of neutralizing monoclonal antibodies has been shown to improve disease outcome for some patients with COVID-19 (Corti et al., 2021). As variants of SARS-CoV-2 emerge (Cameroni et al., 2021;Collier et al., 2021;Mlcochova et al., 2021;Tegally et al., 2020;Walls et al., 2022), it is necessary to evaluate their impact on serum neutralizing activity, as a proxy for vaccine efficacy, in order to inform public health policies worldwide and further vaccine development.
Of the three immunogens tested, only RBD-NP induced detectable serum neutralizing activity post-prime (2 weeks after immunization), in contrast to what we observed with AS03-adjuvanted HexaPro S in NHPs (Arunachalam et al., 2021;Walls et al., 2020a). With RBD-NP-elicited sera at week 2, we observed a 2.0-fold reduction and an equivalent neutralization potency against the Beta and Gamma variants of concern (VOCs) relative to G614 using VeroE6-TMPRSS2 cells, respectively ( Figure 1A; Data S1). Using HEK293T-ACE2 cells, the corresponding decreases were 2.9-and 1.3-fold, respectively ( Figure 1B; Data S1). All three protein immunogens induced robust neutralizing antibody responses 2 weeks after boost. Serum neutralizing activity relative to G614 at week 5 was increased 2.4-fold (Beta) and decreased 1.2-fold (Gamma) in mice immunized with RBD-NP, reduced 1.5-fold (Beta) and increased 2.0-fold (Gamma) for S2P, and reduced 1.2-fold (Beta) and increased 1.4-fold (Gamma) with HexaPro S-elicited sera using VeroE6-TMPRSS2 cells ( Figure 1A; Data S1). These findings were overall consistent with the outcomes of the assays using HEK293T-ACE2 cells ( Figure 1B; Data S1), with the exception of neutralizing antibody responses elicited by HexaPro S, which were reduced 4.9-fold against Beta and 2.8-fold against Gamma relative to G614, indicating that these are not antigen-or cell-typespecific observations. These results are consistent with a recent study showing a smaller than expected dampening of neutralizing activity against VOCs using protein subunit immunogens (Amanat et al., 2021).
Since immunogen dose has an effect on total serum neutralizing responses, we immunized BALB/c mice twice (at weeks 0 and 4) with RBD-NP at five different doses ranging from 1.66 to 0.0001 mg. Serum neutralizing activity was measured with G614, Beta, and Gamma VSV pseudotypes. Similar to above, the sera of mice vaccinated twice with 1.66 mg (RBD antigen dose) had 1.6-and 2.7-fold reductions in neutralization potency against Beta and Gamma, respectively ( Figure 1C; Data S1). Sera elicited by two 0.1 mg vaccine doses had a 2.2-and 2.4-fold respective potency reductions against Beta and Gamma S VSV, whereas two 0.01 mg vaccine doses led to 3.6-and 1.5-fold respective potency reductions against Beta and Gamma S VSV. Mice immunized with two 0.001 mg doses experienced 2.0-and 1.5-fold reductions of serum neutralizing activity against Beta and Gamma S VSV pseudotypes, respectively ( Figure 1C; Data S1). No serum neutralizing activity was observed for the lowest (0.0001 mg) dose, preventing further investigations of potential differences at this dose ( Figure 1C; Data S1). These data show no correlation between vaccine dose and the ratio of neutralizing activity between G614 and VOCs.
To explore the generalizability of these observations, we immunized BALB/c mice twice (at weeks 0 and 3) with Moderna mRNA-1273 (Wu et al., 2021) and collected sera at week 5. Serum neutralizing activity 2 weeks after boost, measured in a different laboratory, was reduced $2-fold against both Beta and Gamma variant pseudoviruses compared with G614 S using A549/ACE2/TMPRSS2 cells (Figure 1D; Data S1) (Wu et al., 2021). To determine whether these findings were specific to BALB/c mice, we immunized K18-hACE2 transgenic and 129S2 mice with mRNA-1273 twice (3 weeks apart) and collected sera at week 6 (Ying et al., 2022). Serum neutralizing activity was evaluated using a focus-reduction neutralization test (FRNT) with authentic SARS-CoV-2 carrying the G614 S glycoprotein in a WA1/2020 background or a clinical isolate of the Beta VOC (Case et al., 2020a). We observed a 1.8-and 1.3-fold increased neutralization potency of the Beta variant compared with G614 for 129S2 and K18-hACE2 immunized mice, respectively (Figure 1E; Data S1). These data indicate that the unexpected apparent resilience of variants to polyclonal neutralizing antibody responses in mice appears to be independent of the mouse model, the vaccine platform, the nature of the antigen, the neutralization assay used, or the target cell type.
To understand whether these observations extend beyond mice, we analyzed sera obtained from pigtail macaques and rhesus macaques immunized with RBD-NP or HexaPro S protein subunit vaccines (Arunachalam et al., 2021;Walls et al., 2020aWalls et al., , 2021. Pigtail macaques (n = 2) were immunized with AddaVaxadjuvanted RBD-NP at weeks 0 and 4, and sera from week 12 were analyzed. Rhesus macaques were immunized at weeks 0 and 3 with RBD-NP adjuvanted with AS37 (n = 5), CpG-alum (n = 5), or alum (n = 5). Rhesus macaques (n = 5) immunized with HexaPro S adjuvanted with AS03 were used to compare RBD-and S-based vaccines. Rhesus macaque sera from week 6 were used for our analysis. All NHP serum neutralizing titers were lower than immunogen-matched mice titers. Serum neutralizing activity from pigtail macaques immunized with AddaVax-adjuvanted RBD-NP showed a 2.7-fold potency dampening against Beta and 4-fold against Gamma VOC compared with G614 S pseudovirus using HEK293T-ACE2 cells ( Figure 1F; Data S1). Rhesus macaques immunized with AS37adjuvanted RBD-NP had a reduction in neutralization potency of R11-fold (Beta) and 8.6-fold (Gamma), whereas a similar $6to 8-fold decrease was detected with CpG-alum and alum for both Beta (8.2 for CpG-alum and 7.2 for alum) and Gamma (7.8 for CpG-alum and 6.4 for alum) using HEK293T-ACE2 cells ( Figure 1F; Data S1). AS03-adjuvanted HexaPro S immunization led to 11.2-and 6.4-fold reductions in serum neutralizing activity against Beta and Gamma S VSV, respectively ( Figure 1F; Data S1). These data recapitulate previous findings made with neutralization of authentic viruses (Arunachalam et al., 2021), suggesting that these outcomes are not specific to pseudovirus assays. Collectively, these data obtained with different clinical adjuvants indicate that vaccine-elicited neutralizing antibody responses in NHPs are significantly more affected by VOCs than those in BALB/c mice.
Analysis of serum neutralizing antibody titers in individuals vaccinated with either Pfizer/BioNTech Comirnaty or Moderna mRNA-1273 confirmed that the magnitudes of potency reduction against the Beta and Gamma variants relative to G614 are on par with our NHP data and that both are much greater than those observed in BALB/c mice ( Figure 1G; Data S1; Table S1). Compared with G614 S, we observed 6.1-fold (Beta) and 2.1-fold (Gamma) reductions in neutralization potency with a cohort of humans immunized with Pfizer/BioNTech Comirnaty and 8.7-fold (Beta) and 3.2-fold (Gamma) reductions with a cohort of humans immunized with Moderna mRNA-1273 using a VSV pseudovirus assay with HEK293T/ACE2 target cells ( Figure 1G; Data S1). These results are in line with the average attenuations of neutralizing antibody responses retrieved from the coronavirus antiviral and resistance database (https:// covdb.stanford.edu/), which are 7.3 (Beta) and 4.1 (Gamma) for Pfizer/BioNTech Comirnaty and 7.8 (Beta) and 4.5 (Gamma) for Moderna mRNA-1273 ( Figure 1H).
We next tested whether using mouse sera obtained 8 weeks post-prime would reveal larger differences between G614 and VOCs. At 8 weeks after RBD-NP prime (5 weeks after boost), mouse serum neutralizing activity remained mildly affected when comparing G614 to either Beta or Gamma S VSV pseudotyped viruses in VeroE6/TMPRSS2 cells ( Figure 1A; Data S1). A mild attenuation (3.3-fold) of serum neutralizing activity was also observed 8 weeks post-prime against the Beta variant with a cohort of 104 BALB/c mice immunized with mRNA-1273 ( Figure 1D; Data S1). In contrast, HexaPro S-immunized mice exhibited a 5.9-fold drop of serum neutralizing activity against Beta and 1.3-fold against Gamma S VSV pseudotyped viruses (Figure 1A; Data S1), more closely resembling the expected trends for NHP and human data ( Figures 1F and 1G; Data S1). Vaccine-elicited BALB/c mice sera post-prime and after boost did not recapitulate observations made with NHPs and humans regarding the impact of SARS-CoV-2 variants on neutralizing activity regardless of overall titer, dose, time following vaccination, vaccine modality, target cell type, or antigen.

DISCUSSION
Although BALB/c and other mice are widely used for evaluating vaccine candidates, our data suggest that they are not always an adequate animal model to evaluate the breadth and potency of neutralizing antibody responses against emerging SARS-CoV-2 VOCs, potentially due to their different immune repertoires compared with primates. For example, polyclonal neutralizing antibody responses resulting from infection or vaccination with ancestral SARS-CoV-2 isolates in humans and NHPs can be hyperfocused on position 484 in the RBD, which does not appear to be recapitulated in mice (Greaney et al., 2021;Walls et al., 2021). However, intranasal delivery of an S2P-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in K18-hACE2 transgenic mice (Chen et al., 2021;Hassan et al., 2021) and intramuscular delivery of SARS-CoV-2 S mRNA-LNP to BALB/c mice (Martinez et al., 2021) show fold changes in line with the expected dampened human responses for Beta compared with G614, suggesting there could be exceptions to the generalizability of these findings. Further experiments will be necessary to understand the immunological basis of these findings, such as comparing vaccine-elicited responses in animals of both sexes, inbred versus outbred mouse strains (Gralinski et al., 2015), or exploring the microbial environment of the mice (Fiege et al., 2021). Another alternative may be to utilize humanized mice (Lee et al., 2014;Murphy et al., 2014;Nie et al., 2021;Walls et al., 2020a;Wang et al., 2020) to better match the antibody repertoire of primates as our data suggest that other animal models may be better suited to evaluate serum immune evasion of new variants as they emerge, even though mice are a cost-and time-effective option for evaluating overall immunogenicity.

Limitations of the study
This study is an observation of the dampening of neutralizing activity toward SARS-CoV-2 variants in mice compared with NHPs or humans. This study focused on characterizing the breadth and robust nature of these observations and not on the mechanisms behind these distinct differences in mice such as antibody repertoire, animal sex, inbred versus outbred mice, the microbial environment, etc., which will be important questions for future studies. Another limitation of this study is the low animal subject number, in some cases having an n of 2, and the study could be enhanced by larger numbers of animals within each vaccine type.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, David Veesler (dveesler@uw.edu).

Materials availability
Materials generated in this study will be made available on request after signing a materials transfer agreement with the University of Washington. REAGENT

Data and code availability
Data generated in this study are presented in Data S1 and data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.  , 2021). The A549-hACE2-TMPRSS2 was produced from adenocarcinomic human alveolar basal epithelial cells.All adherent cells were cultured at 37 C with 5% CO 2 in flasks with DMEM +10% FBS (Hyclone) + 1% penicillin-streptomycin. Cell lines were not tested for mycoplasma contamination nor authenticated.

Transient transfection
Proteins were produced using endotoxin free DNA in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33 C, 70% humidity, 8% CO 2 rotating at 150 rpm. The cultures were transfected using PEI-MAX (Polyscience) with cells grown to a density of 3.0 million cells per mL and cultivated for 3 days. Supernatants were clarified by centrifugation (5 min at 4000 rcf), addition of PDADMAC solution to a final concentration of 0.0375% (Sigma Aldrich, #409014), and a second spin (5 min at 4000 rcf).

Protein purification
Proteins containing His tags were purified from clarified supernatants via a batch bind method where each clarified supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of $310 mM. Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 min with gentle shaking. Resin was collected using vacuum filtration with a 0.2 mm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with 3 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. The batch bind process was then repeated and the first and second elutions combined. SDS-PAGE was used to assess purity. RBD-I53-50A fusion protein IMAC elutions were concentrated to >1 mg/mL and subjected to three rounds of dialysis into 50 mM Tris pH 7.4,