The SARS-CoV-2 Omicron subvariant BA.5 rapidly became globally dominant due to its unique spike protein mutations mediating immune escape and increased ACE2 receptor binding. To address concerns about vaccine efficacy against BA.5, many countries adopted booster dose strategies. This study uses male Syrian hamsters to comprehensively evaluate neutralizing antibody responses and efficacy against BA.5 challenge after various prime/boost vaccination regimens using Ad26.COV2.S (Janssen), BNT162b2 (Pfizer/BioNTech), mRNA-1273 (Moderna), and NVX-CoV2373 (Novavax). The study aims to provide insights into optimal COVID-19 vaccine strategies and inform human vaccination recommendations. The global effort to rapidly develop and deploy COVID-19 vaccines, utilizing various platforms including viral vectors, mRNA, and protein subunits, has significantly reduced infections, hospitalizations, and deaths. However, emerging variants like Alpha, Delta, and Omicron, with spike protein mutations causing immune escape, necessitate booster strategies to maintain protection. This study focuses on BA.5, characterized by mutations leading to increased reinfections and breakthrough cases, to assess the efficacy of different vaccination approaches in an animal model.

Several studies have shown the effectiveness of COVID-19 vaccines in reducing severe disease and death. However, the emergence of new variants, especially Omicron subvariants, has raised concerns about the long-term efficacy of existing vaccines. Prior research has examined the immune response to various vaccine platforms, including mRNA vaccines (BNT162b2, mRNA-1273) and viral vector vaccines (Ad26.COV2.S). Studies have shown that these vaccines induce neutralizing antibody responses, but the durability of this protection and the effectiveness against emerging variants have been areas of ongoing investigation. The immune escape properties of BA.5 have been documented, highlighting the need for effective booster strategies. Research has also explored heterologous boosting, using different vaccine platforms for the primary series and booster, to potentially enhance immune responses. Previous animal studies have shown the potential of homologous and heterologous boosters in improving protection against earlier SARS-CoV-2 variants, laying the groundwork for this investigation into the BA.5 subvariant. Specifically, studies have shown effectiveness of NVX-CoV2373, a protein-based vaccine, in inducing neutralizing antibodies in humans, which is explored in this study as a potential booster candidate.

This study used four-to-five-week-old male Syrian hamsters. One group received a single dose of 10¹⁰ or 10⁸ viral particles (vp) of Ad26.COV2.S, another group received two doses of 5 µg of BNT162b2 four weeks apart, and a control group received PBS. Neutralizing (nAbs) and binding (bAbs) antibody responses against the ancestral SARS-CoV-2 strain were measured using plaque reduction neutralization tests (PRNT) and enzyme-linked immunosorbent assays (ELISA). Six months after the initial immunization, hamsters received a homologous booster (Ad26.COV2.S or BNT162b2) or a heterologous booster (mRNA-1273 or NVX-CoV2373). Neutralizing antibody activity against BA.5 was evaluated at various time points. Three months post-boost, hamsters were challenged with 10⁴ PFU of Omicron BA.5. Infectious viral titers were measured in nasal washes, trachea, and lungs. Daily weights and signs of disease were recorded. Histopathological evaluation of lung tissues was performed. The correlation between neutralizing antibody titers and protection against BA.5 was analyzed using Spearman's rank correlation coefficient. Statistical analyses included one-way ANOVA, Mann-Whitney test, and Wilcoxon signed-rank test. ELISAs were used to measure binding antibodies, and PRNT and FRNT assays measured neutralizing antibodies. Viral titers were determined by plaque assays. Histological analysis evaluated lung tissue damage. Statistical analysis employed one-way ANOVA, Mann-Whitney, and Wilcoxon tests depending on data distribution.

The study found that a single dose of Ad26.COV2.S (at either high or low dose) induced a more durable neutralizing antibody response compared to two doses of BNT162b2. Importantly, the heterologous boost with NVX-CoV2373 resulted in a substantial increase in neutralizing antibodies against BA.5 and provided the strongest protection against BA.5 infection. One month post-boost, NVX-CoV2373 boosted animals showed a 2.6-3.8-fold increase in neutralizing antibodies compared to pre-boost levels, depending on the primary vaccine. Three months post-boost, this increase was maintained. The NVX-CoV2373 booster resulted in significantly less weight loss following BA.5 challenge compared to sham-vaccinated controls. Hamsters boosted with NVX-CoV2373 showed the highest level of protection against BA.5 infection, with significant reductions in viral load in nasal washes, trachea, and lungs compared to sham-vaccinated animals. Histopathological analysis showed that NVX-CoV2373 boosted animals had milder lung lesions. A strong inverse correlation was observed between pre-challenge neutralizing antibody titers and viral loads in the lungs at 4 dpi, particularly in animals boosted with NVX-CoV2373. A detectable neutralizing antibody titer (FRNT₅₀≥20) appeared to completely prevent lung infection at 4 dpi. Sham- or homologous-boosted hamsters showed the highest proportion of breakthrough infections.

The findings demonstrate that a heterologous boosting strategy, particularly using NVX-CoV2373, significantly enhances protection against the Omicron BA.5 subvariant in a Syrian hamster model. The more durable immune response induced by Ad26.COV2.S compared to BNT162b2 highlights potential advantages of different vaccine platforms. The strong correlation between neutralizing antibody titers and protection underscores the importance of humoral immunity in combating BA.5 infection. The superior performance of NVX-CoV2373 as a booster suggests its potential as a valuable tool in COVID-19 vaccination strategies. These results have significant implications for informing public health decisions regarding booster dose recommendations and vaccine selection.

This study provides strong evidence supporting the use of heterologous boosting, particularly with NVX-CoV2373, to enhance protection against SARS-CoV-2 Omicron subvariant BA.5. The superior performance of NVX-CoV2373 should be considered in future vaccination strategies. Future research should investigate the efficacy of these vaccine combinations against newer Omicron subvariants and explore the role of cellular immunity in overall protection. The durability of the Ad26.COV2.S-induced immune response warrants further investigation.

The study was conducted exclusively in male Syrian hamsters, limiting the generalizability to other animal models and human populations. The study primarily focused on humoral immunity, neglecting potential contributions of cellular immunity. The use of a single concentration of NVX-CoV2373 might have led to an overestimation of protection. The low dose of mRNA-1273 used may not be directly comparable to optimal doses in humans. The challenge was performed only with BA.5, and results may not extrapolate to other circulating variants. The high doses used in the rodent model may not be fully comparable to human doses.