Parental/Beta bivalent CoV2 preS dTM-AS03 confers balanced and broader neutralizing antibody responses than D614 or Beta monovalent vaccines in naïve NHPs
We first checked for potential immune interference between the two valences in the bivalent formulation as compared to the D614 or Beta monovalent formulations. Naïve cynomolgus macaques were immunized on D0 and D21 with the bivalent CoV2 preS dTM-AS03 (D614 + Beta) vaccine formulation at 5 µg + 5 µg dose, or monovalent CoV2 preS dTM-AS03 vaccines (D614 or Beta) at 5 µg and 10 µg doses (Fig. 1).
Neutralizing antibody (nAb) responses against the parental Wuhan strain D614 and the Beta VOC were analyzed two weeks post-dose 2 using lentivirus based pseudovirus assays (lentivirus-PsV). The bivalent vaccine candidate elicited high and consistent nAb titers against parental SARS-CoV-2 D614, with mean titers of 3.0 log10 ± 0.4 log10 standard deviation (SD) (Supplementary Fig. 1a), not significantly different from those induced by the monovalent D614 at 5µg (mean titers of 3.1 log10) and 10µg (mean titers of 3.4 log10), but significantly higher than those elicited by the monovalent Beta vaccine at 10µg (mean titers of 2.3 log10, P = 0.0017, mean fold increase 4.9). The absence of negative immune interferences in the bivalent formulation was confirmed using a qualified D614 vesicular stomatitis virus (VSV)-pseudovirus assay (Supplementary Fig. 1b).
As expected, the bivalent vaccine also elicited high and consistent nAb titers against Beta pseudovirus in all macaques, with a mean of 3.0 log10 ± 0.3 log10 SD, equivalent to those induced by the monovalent Beta vaccine at 5 µg and 10 µg (Supplementary Fig. 1c) and significantly higher than those induced by the monovalent D614 at 10µg (7-fold mean increase, P <0.001).
Overall, the bivalent CoV2 preS dTM-AS03 induced robust and balanced nAb titers against both D614 parental strain and Beta variant, with no detectable immune interferences.
We next assessed the breadth of neutralization conferred by the bivalent vaccine against other VOC, D614G, Alpha and Delta (all containing the original E484 position), Gamma and Mu (containing the E484K mutation present in Beta) and the more distant Omicron BA.1 (containing E484A mutation among 15 mutations in the RBD) and SARS-CoV-1. The bivalent vaccine was compared to the monovalent vaccine formulations at the same time point (i.e. 2 weeks post second dose) (Fig. 2a).
The bivalent vaccine elicited high and homogenous nAb titers against all VOC - except Omicron BA.1 - with mean titers ranging from 2.8 log10 against Delta to 3.5 log10 against Gamma. When compared to the monovalent D614 vaccine (10µg), the bivalent vaccine elicited equivalent nAb titers against D614, D614G, Alpha and Delta (mean titers of 3 to 3.5 log10), but higher nAb titers against Beta, Gamma and Mu (mean titers of 2.2 to 2.4 log10, P < 0.001, mean fold-increase from 6.1 to 12.2). Conversely, compared to the Beta monovalent, the bivalent vaccine induced higher nAb titers against D614, D614G, Alpha and Delta (mean titers of 2.1 to 2.5 log10, mean fold increase from 3.3 to 5.8, P < 0.05) but equivalent nAb titers against Beta, Gamma and Mu (mean titers of 2.9 to 3.3 log10).
For Omicron and SARS-CoV-1, the bivalent vaccine induced detectable titers in all animals, with mean titers around 2.0 log10, whereas nAb responses were more variable in the monovalent vaccine groups.
The results indicate that the parental/Beta bivalent vaccine candidate elicits robust and balanced nAb responses against the original E484-containing viruses (D614, D614G, Alpha and Delta), the E484K-containing viruses (Beta, Gamma and Mu) and lower but consistent nAb responses against the distant Omicron (E484A) and SARS-CoV-1.
For comparison, the bivalent vaccine elicited mean D614 nAb titers higher than that measured in the World Health Organization (WHO) International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC code: 20/136; 2.8 log10 in our lentivirus-pseudovirus assay) and higher than those measured in a panel of 93 human convalescent sera (collected within 3 months after positive PCR test; 2.0 log10; Fig. 2b)
Two-dose primary series with the parental/Beta bivalent vaccine induces more balanced and more homogeneous nAb titers than a heterologous prime/boost regimen in naïve NHPs
To assess the benefit of heterologous prime/boost immunization on cross-neutralization, two alternative immunization regimens were tested, where a first dose with the monovalent D614 (D0) was followed by a second dose (D21) with either the monovalent Beta or the bivalent vaccines (Supplementary Fig. 2a).
Among the two heterologous prime/boost regimens, that with the monovalent Beta used for the second injection was able to extend the breadth of the neutralizing response to Beta and other VOC, although with higher variability and slightly lower titers than the bivalent vaccine 2-dose regimen (not significant for D614, D614G, Alpha, and Delta mean nAb titers, 3- and 4.1-fold lower for Beta, p=0.0165 and Gamma, P = 0.0052 mean nAb titers, respectively) (Supplementary Fig. 2b). When the second injection was performed with the bivalent vaccine, the breadth of cross-neutralization was not different from that observed with the D614 monovalent 2-dose regimen (Fig. 2 and Supplementary Fig. 2b).
The parental/Beta bivalent vaccine elicits durable nAb titers and memory B cell responses in naïve NHPs
To address potential neutralizing Ab waning, we measured the nAb titers against the parental D614G, Beta, Delta and Omicron in the blood of vaccinated animals up to 6-months post-immunization (D206) with the bivalent and monovalent vaccine formulations at 10 µg total antigen dose (Fig. 3).
For each vaccine formulations, nAb titers peaked on D34, decreased up to D70 or D114 and then remained stable up to 6 months (D206). All animals had detectable PsV nAb titers up to 6 months, except against Omicron in the monovalent D614 vaccine group (Fig. 3d). The parental D614G and Beta variant mean nAb titers at 6 months were, respectively, 2.6 and 2.4 log10 for the bivalent vaccine groups, 2.7 and 1.8 log10 for the D614 monovalent, 2.2 and 2.5 log10 for the Beta monovalent. The Delta and Omicron mean nAb titers at 6 months were, respectively, 2.1 and 2.0 log10 for the bivalent vaccine groups, 2.2 and 1.5 log10 for the D614 monovalent, 1.8 and 2.0 log10 for the Beta monovalent. The Alpha and Gamma mean nAb titers at 3 months (D114) were, respectively, 2.6 and 2.6 log10 for the bivalent vaccine groups, 2.7 and 2.1 log10 for the D614 monovalent, 2.3 and 2.8 log10 for the Beta monovalent (Supplementary Fig. 3).
Interestingly, the decay appeared less pronounced for cross-neutralizing responses than for vaccine-homologous neutralizing responses. Of note, the cross-neutralizing responses tended to peak at lower titers than vaccine-homologous neutralizing responses (Fig. 3, Supplementary Fig. 3). Thus, the difference in cross-neutralizing titers were less at 6 months post-immunization than at 2 weeks post second dose. This holds especially for Omicron for which no significant decline was observed with the bivalent vaccine between D34 and 6 months nAb titers.
Modelling the Ab decay based on the experimental data indicated that, after the initial decay, parental D614G nAb titers reached a plateau in the monovalent D614 10µg group on D93, with mean titer estimated at 2.8 log10. In the bivalent group, the plateau was not detected but the decay was very low (0.3 log10/100 days), and in the monovalent Beta 10µg group, the decay was not significant. Beta nAb titers reached a plateau for all groups, estimated at 2.4 log10 on D118, 1.8 log10 on D113, 2.5 log10 on D119, and for bivalent, monovalent D614 and monovalent Beta, respectively.
On D114, S-specific IgG-secreting memory B cell responses were detected for all animals at similar levels across all three vaccine groups i.e. bivalent, monovalent D614 and monovalent Beta (Fig. 4). When adjusted to total IgG memory B cells, the responses were very homogeneous within groups. The medians were 0.15, 0.32 and 0.37% of S-specific/total IgG-secreting memory B cells for the bivalent, monovalent D614 and monovalent Beta vaccine groups, respectively.
Parental/Beta bivalent vaccine induces similar antibody Fc functions as monovalent vaccines
In addition to neutralizing antibodies, Fc receptor-binding and antibody effector functions were assessed as they have been previously shown to contribute to the clearance of SARS-CoV-2 infection29. The bivalent formulation elicited similar IgG1 titer and FcR2a-binding antibodies against D614 and Beta Spikes compared to the monovalent vaccine formulations at 5 µg and 10 µg (Fig. 5a-b).
We then measured the ability of the antibodies from these different vaccine formulations to induce cellular effector functions. We found that the bivalent formulation induced significantly less antibody-dependent cellular phagocytosis (ADCP) against D614 Spike compared to the monovalent formulation at 10 µg (Fig. 5c). No significant differences were observed for the ability of the antibodies to elicit antibody-dependent complement deposition (ADCD) (Fig. 5D).
To understand the difference between the bivalent and monovalent formulations on a broad-level, we plotted a heatmap of the z-score of all measured antibody titers, FcR-binding and antibody effector functions against D614 (Fig. 5e, left) or Beta (Fig. 5e, right). This analysis did not reveal any specific pattern that discriminate the bivalent from the monovalent formulations. These data highlight that the bivalent formulation induces robust IgG subclass antibody titer and FcR-binding function, comparable to the monovalent formulations.
Further analyses of the effector functions induced by the different formulations were performed against Omicron variant (BA.1) spike. The IgG1 binding titer to the Omicron spike was highly correlated to the IgG1 D614G binding (Supplementary Fig. 4a), but showed a decreased binding as shown in previous studies30. The bivalent formulation induced Spike-specific antibodies against Omicron with similar titers (Supplementary. Fig. 4b) and similar FcR2a and FcR3a binding (Supplementary Fig. 4c) compared to the monovalent formulations D614 and Beta at 5 µg and 10 µg. Whereas all formulations induced similar antibody-dependent cellular and neutrophil phagocytosis (ADCP and ADNP, respectively), the 10 µg Beta monovalent formulation induced higher ADCD than the 5 µg Beta monovalent formulation, and trended higher than all other formulations (Supplementary Fig. 4d). Whether the increase in ADCD activity is simply due to higher vaccine dose or the induction of IgM against Omicron is unclear.
Finally, to understand whether the bivalent vaccine formulation induces a wider breadth of antibody response, we plotted the median percentile rank of the IgG1 titers for the bivalent formulation and the four monovalent formulations against D614G, Omicron, Alpha, Beta, and Delta Spike (Supplementary Fig 4e). This analysis suggests that the bivalent formulation and the two monovalent D614 and Beta formulations at 10 µg may offer a broader IgG response than the monovalent formulations at 5 µg.
The parental/Beta bivalent vaccine formulation induces robust and balanced neutralizing antibody responses against parental SARS-CoV-2 and VOC in hamsters
We next investigated the protection conferred by the bivalent vaccine in Golden Syrian Hamsters against viral replication and lung pathology induced by viral challenge with the parental D614G, Alpha and Beta variants. Three cohorts of 32 hamsters (each divided in 4 groups of 8 animals) were immunized with either, buffer (control group), monovalent D614, monovalent Beta or bivalent (D614 + Beta) CoV2 preS dTM-AS03, using the 2-dose D0/D21 regimen, by intramuscular route at 1µg of each antigen (Fig. 6a). Four weeks post second dose, each cohort was challenged with either, the parental D614G, Alpha or Beta viruses, using infectious doses previously characterized to induce between 10 and 20% body weight loss.
Before challenge, S-specific IgG (ELISA) and nAb titers against parental D614G, Alpha, Beta and Delta VOC were evaluated in sera from vaccinated and non-vaccinated hamsters, using lentivirus-pseudovirus assays. The different vaccine formulations induced S-specific IgGs in all hamsters 21 days after the first injection except for 1 and 2 hamsters in the D614 and Beta monovalent vaccine groups, respectively. Mean IgG titers varied from 3.2 to 3.5 log10 ELISA Units (EU) as shown in Fig. 6b and were comparable in all the vaccine groups. The mean S-specific IgG titers increased to ~5.0 log10 EU, 2 weeks post second dose, with no statistically significant differences between the different vaccine formulations. Consistent with the prior observations in macaques, the bivalent vaccine formulation elicited high and balanced nAb titers at D35 against parental D614G, Alpha, Delta and Beta with mean titers between 2.7 to 3.2 log10 (Fig. 6c). The monovalent D614 formulation induced high and balanced nAb titers against parental D614G, Alpha and Delta (mean titers between 2.9 and 3.1 log10), whereas nAbs were lower against Beta (mean titers of 1.5 log10). It is noteworthy that two hamsters vaccinated with the monovalent D614 vaccine had low S-specific IgG titers (3.0 and 4.2 log10 EU) and no detectable nAb titers, 2 weeks post second dose. Conversely, the monovalent Beta formulation induced only high nAb titers against the homologous Beta pseudovirus (mean titers of 3.1 log10) and lower nAb titers against parental D614G, Alpha and Delta (mean titers between 1.9 and 2.5 log10; Fig. 6c). Those data are in line with our observations in macaques showing no immune interferences between the two antigens.
Statistically significant higher nAb titers against Beta were observed with the bivalent formulation compared to the monovalent D614 formulation (38-fold, P < 0.001), and for the parental D614G (6.5-fold, P < 0.001), Alpha (3.7-fold, P = 0.0144) and Delta (7.3-fold, P < 0.001) nAb titers compared to the monovalent Beta vaccine.
The parental/Beta bivalent vaccine confers protection against D614G, Alpha and Beta variants replication and pathology in hamsters
To assess protection against parental (D614G) and variants (Alpha and Beta), body weight change was monitored up to 7 days post-challenge as a marker of disease progression, and lung viral loads and pathology were assessed after necropsy on D4 or D7 on half the animals. Unvaccinated hamsters (buffer) lost up to 18% of body weight 7 days post-challenge with Beta and Alpha and up to 10% after parental D614G challenge (Fig. 7a), while non-challenged hamster weights remained stable or slightly increased during the same period (data not shown). Except for the two low responder hamsters (with no nAb titers) vaccinated with D614 monovalent vaccine, all vaccinated hamsters, whatever the vaccine formulations, had stable or slightly increasing body weight post-challenge, following the same profile as the non-challenged group (data not shown).
Viral loads were measured in lungs 4- and 7-days post-challenge (half of the animals per group and per timepoint) using 50% Tissue Culture Infectious Dose (TCID50) titration. In each cohort, the unvaccinated group (buffer) showed high viral loads 4 days post challenge at mean viral loads of 8.8 log10, 8.6 log10, and 8.3 log10 TCID50 in the D614G, Alpha and Beta cohorts respectively (Fig. 7b). On D4 post-challenge, hamsters vaccinated with the bivalent or the monovalent Beta vaccines displayed low to undetectable viral loads, irrespectively of the virus used for the challenge (D614G, Alpha or Beta). Hamsters vaccinated with the monovalent D614 vaccine also displayed low to undetectable viral loads on D4, except for the two hamsters in the Alpha challenge cohort that showed high viral loads consistent with body weight loss and no detectable nAb titers. One hamster vaccinated with the monovalent D614 also showed high viral load in the cohort challenged with the Beta variant.
In the three cohorts, infectious titers were low to undetectable 7 days post-challenge.
We next assessed lung pathology 4- and 7-days post-challenge by microscopic evaluation of lung sections stained with hematoxylin and eosin (H&E) or subjected to anti-SARS-CoV-2 nucleoprotein immunohistochemistry (IHC). For all three viral strains, pathology in unvaccinated hamsters (buffer) consisted of multifocal to coalescing regions of inflammatory infiltrate composed of macrophages, lymphocytes, heterophils and syncytial cells admixed with hemorrhage and cellular debris often obliterating normal architecture (Fig. 8a, Supplementary Fig. 5). There was marked type II pneumocyte hyperplasia and bronchiolar epithelium was multifocally hyperplastic characterized by many epithelial cells piling up and interspersed with rare necrotic cells. The lumena of some bronchioles contained necrotic cellular debris. Blood vessels reveal either mural or perivascular infiltration by mixed inflammatory infiltrates. Lung pathology was markedly attenuated in hamsters that received vaccination. Semi-quantitative scores were made based on the percent of lung affected. Lungs that were consistent with normal tissue were given a score of 0. If less than 25% of the section revealed histopathology, a score of 1 was assigned. A score of 2 was attributed to lung sections in which histopathology was present in greater than 25% but less than 50% of the lung. If more than 50% of the lung parenchyma was involved, a score of 3 was assigned.
All unvaccinated hamsters had median lung pathology scores of 2 (on a scale of 0-3), 4 days post-challenge with parental D614G and Alpha, and a score of 1 or 2 in the group challenged with Beta (Fig. 7c). Seven days post-challenge, the pathology progressed in all unvaccinated animals to the maximal score of 3, irrespective of the strain used for the challenge. In contrast, hamsters vaccinated with the monovalent or bivalent vaccines displayed no or minimal pathology (scores of 0 or 1), at day 4 or 7 post-challenge, except for the two low responder hamsters vaccinated with D614 and challenged with the Alpha variant which displayed scores of 2 and 3, consistent with the high viral loads. In the same cohort (Alpha challenge), one hamster vaccinated with the monovalent Beta showed a score of 2, 4 days post challenge, however post-hoc analysis indicated that the pathology was less developed than in the buffer immunized controls with similar scores. Moreover, while immunohistochemistry showed Nucleocapsid protein expression in multifocal to coalescing regions of the lungs in unvaccinated hamsters 4 days post challenge, nucleocapsid protein was not detected in any vaccinated hamsters at the same timepoint, except in the two low responder animals immunized with the monovalent D614 vaccine (Fig. 7d, Fig. 8b).
Overall, the D614/Beta bivalent as well as the two monovalent CoV2 preS dTM-AS03 (D614 and Beta) vaccines conferred protection in hamsters against body weight loss, viral replication in lungs and lung pathology induced by parental D614G, Alpha or Beta variants.