PROTECTIVE IMMUNITY AGAINST GX_P2V(short_3UTR) IN GOLDEN HAMSTERS IMMUNIZED WITH VIRAL ITSELF
We initiated our study by evaluating the immunogenicity of GX_P2V(short_3UTR) in golden hamsters. Intriguingly, a single immunization resulted in long-term protective immunity against the virus. Notably, robust, sterile immunity was realized after a second immunization. Eighteen golden hamsters were divided into three distinct groups: mock, single-immunization, and double-immunization (Fig. 1a). Both immunizations and subsequent challenges were delivered intranasally with a dose of 1×105 TCID50, separated by a 21-day interval. Post the single and double immunizations, challenge infections were administered after another 21-day period. Subsequently, we assayed the viral RNA and titers in lung and tracheal tissues on days 3 and 5 post-infection (Fig. 1b-e). Remarkably, neither viral RNA nor titers were detectable in the lung tissues after both single and double immunizations (Fig. 1b, d). Conversely, low viral RNA loads were observed in tracheal tissues on days 3 and 5 post-challenge after a single immunization, but were absent following a double immunization (Fig. 1c). This trend persisted when the challenge was carried out three months after a single immunization, with low viral RNA loads in both lung and tracheal tissues, but absent viral titers (Fig. 1f-h). Similarly, when golden hamsters were challenged 2 years later, low levels of viral RNA were detected in the lungs and trachea on day 3 post-infection despite lack of viral titers (Fig, 1i-k). These observations confirm that while a single dose of GX_P2V(short_3UTR) induces sustained protective immunity, two doses are requisite for optimal sterile immunity. Moreover, the protective outcomes post-single immunization align with reported immune responses observed after recovery from a single SARS-CoV-2 infection in golden hamsters6, suggesting the potential of enhanced immunity upon coronavirus reinfection.
PROTECTIVE EFFICACY OF GX_P2V(short_3UTR) IMMUNIZATIONS AGAINST SARS-CoV-2 IN GOLDEN HAMSTERS
We proceeded to assess if double immunization with GX_P2V(short_3UTR) could protect golden hamsters from significant weight loss following wild-type SARS-CoV-2 infection. Ten six-week-old golden hamsters were assigned into two cohorts: a mock control and a double immunization group, with doses administered two weeks apart (Fig. 2a). Forty days post a second immunization, all animals were challenged with 2×105 PFU of wild-type SARS-CoV-2, with daily weight monitoring thereafter. By day seven post-infection, control hamsters exhibited a 10% weight reduction, while double-immunized hamsters began gaining weight from day two (Fig. 2b). Considering that SARS-CoV-2-induced pneumonia often correlates with early weight loss in these hamsters6,7, our data suggests that GX_P2V(short_3UTR) double immunization effectively mitigates disease severity.
In a subsequent experiment, hamsters doubly immunized with GX_P2V(short_3UTR) were challenged with SARS-CoV-2 132 days post-immunization to discern long-term immunity (Fig. 2c). Comparison was made against a mock immunized control and a three-dose group (a booster administered 90 days post-second dose). Lung, tracheal, and turbinate tissues were harvested on the 3rd and 5th days post-challenge for viral RNA quantification (Fig. 2d-f). On day three, relative to the mock group, viral RNA in the lung tissues diminished 28-fold with two doses and 187-fold with three doses. By day five, reductions reached 13,760-fold and 65,954-fold, respectively (Fig. 2d). In the trachea, reductions of 9-fold and 22-fold were observed on day three, while by day five, differences became insignificant (Fig. 2e). Turbinate tissues showed 4-fold and 10-fold reductions on day three, and 5-fold and 53-fold reductions on day five for the double and triple doses respectively (Fig. 2f). Notably, the three-dose regimen achieved a more profound viral load decrease. Post-challenge histopathological examination of lung tissues revealed typical viral interstitial pneumonia features, such as alveolar wall thickening and significant mononuclear inflammatory cell infiltration in control hamsters (Fig. 2g)6,7. Conversely, hamsters from both the double and triple immunization cohorts exhibited minimal pathological alterations (Fig. 2g). In summary, double immunization with GX_P2V(short_3UTR) elicits durable protective immunity against SARS-CoV-2 in golden hamsters.
CROSS-NEUTRALIZATION AGAINST GX_P2V(Short_3UTR) AND XBB.1.16 IN CONVALESCENT SERA OF XBB.1.16, AND A NOVEL IMMUNE EVASION STRATEGY OF XBB.1.16
The above wild-type SARS-CoV-2 challenge experiments in golden hamsters corroborate our prior findings that serum from GX_P2V(short_3UTR)-immunized subjects effectively neutralizes the wild-type SARS-CoV-2 pseudovirus4. This prompts an inquiry: can GX_P2V(short_3UTR) elicit neutralizing antibodies with broad reactivity against diverse SARS-CoV-2 variants? Dominant circulating SARS-CoV-2 variants currently include the Omicron strain and its sublineage XBB, both of which exhibit pronounced RBD divergence from GX_P2V(short_3UTR) (Fig. 2h). Here we show that sera from immunized hamsters have variable neutralizing activities against XBB.1.16 (GMT = 26) (Fig. 2j). Nonetheless, we have previously shown that breakthrough infections of the Omicron BF.7 variant in individuals vaccinated against wild-type SARS-CoV-2 yield high-level antibodies cross-neutralizing GX_P2V(short_3UTR), implying the existence of shared neutralizing epitopes5. Extending this, we now demonstrate that XBB.1.16 breakthrough infections in vaccinated individuals induce potent immunity against GX_P2V(short_3UTR) (GMT = 1036) (Fig. 2i), underscoring shared neutralizing epitopes between this variant and GX_P2V(short_3UTR). Serum from unvaccinated individuals convalescing from Omicron BF.7 and XBB.1.16 infections harbor cross-reactive antibodies against GX_P2V(short_3UTR) with geometric mean titer (GMT) values of 49 and 34, respectively (Fig. 2i). Remarkably, neutralization assays using sera from both vaccinated and unvaccinated XBB.1.16 convalescents against XBB.1.16 revealed comparable titers (173.9 vs 126.8) which, intriguingly, were markedly lower than the titer against GX_P2V(short_3UTR) in vaccinated, not unvaccinated, XBB.1.16 convalescents (Fig. 2i-k, Supplementary Table S1-3). These results imply that XBB.1.16 has evolved a decoy-like immune evasion strategy by which high-level neutralizing antibodies against GX_P2V(short_3UTR) induced by epitopes of XBB.1.16 cannot efficiently neutralize XBB.1.16 itself.