mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant

The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

Variable loss of antibody potency against SARS-CoV-2 B.1.1.529 (Omicron)

The recently-emerged SARS-CoV-2 B.1.1.529 variant (Omicron) is spreading rapidly in many countries, with a spike that is highly diverged from the pandemic founder, raising fears that it may evade neutralizing antibody responses. We cloned the Omicron spike from a diagnostic sample which allowed us to rapidly establish an Omicron pseudotyped virus neutralization assay, sharing initial neutralization results only 13 days after the variant was first reported to the WHO, 8 days after receiving the sample. Here we show that Omicron is substantially resistant to neutralization by several monoclonal antibodies that form part of clinical cocktails. Further, we find neutralizing antibody responses in pooled reference sera sampled shortly after infection or vaccination are substantially less potent against Omicron, with neutralizing antibody titers reduced by up to 45 fold compared to those for the pandemic founder. Similarly, in a cohort of convalescent sera prior to vaccination, neutralization of Omicron was low to undetectable. However, in recent samples from two cohorts from Stockholm, Sweden, antibody responses capable of cross-neutralizing Omicron were prevalent. Sera from infected-then-vaccinated healthcare workers exhibited robust cross-neutralization of Omicron, with an average potency reduction of only 5-fold relative to the pandemic founder variant, and some donors showing no loss at all. A similar pattern was observed in randomly sampled recent blood donors, with an average 7-fold loss of potency. Both cohorts showed substantial between-donor heterogeneity in their ability to neutralize Omicron. Together, these data highlight the extensive but incomplete evasion of neutralizing antibody responses by the Omicron variant, and suggest that increasing the magnitude of neutralizing antibody responses by boosting with unmodified vaccines may suffice to raise titers to levels that are protective.

Homogeneous Surrogate Virus Neutralization Assay to Rapidly Assess Neutralization Activity of Anti-SARS-CoV-2 Antibodies

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a ‘Serological Assay based on a Tri-part split-NanoLuc® (SATiN)’ to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Herein, we expand on our previous work and describe a reconfigured version of the SATiN assay that can measure neutralization activity of antibodies directly from convalescent or vaccinated sera. The sensitivity is comparable to cell-based pseudovirus neutralization assays but with significantly shorter preparation and assay run time. As the assay is modular, we further demonstrate that Neutralization SATiN (Neu-SATiN) enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection

The emergence of the Omicron variant (1) of SARS-CoV-2 in November 2021 in South Africa has raised concerns that, based on the large number of mutations in the spike protein and elsewhere on the virus (https://covdb.stanford.edu/page/mutation-viewer/#sec_b-1-351), this variant will have considerable escape from vaccine elicited immunity. Furthermore, several mutations in the receptor binding domain and S2 are predicted to impact transmissibility and affinity for ACE-2. Here we investigated whether Omicron escapes antibody neutralization elicited by the Pfizer BNT162b2 mRNA vaccine and whether the virus still requires binding to the ACE2 receptor to infect cells. We used an early passage of isolated and sequence confirmed live Omicron virus isolated in South Africa. We used a human lung cell line clone (H1299-ACE2) engineered to express the ACE2 receptor (2) to both isolate the virus and test neutralization. We also tested growth in the parental H1299 which do not overexpress ACE2 and are not appreciably infectable with SARS-CoV-2 (Fig S1). The H1299-ACE2 cells were similar to Vero-E6 in titer dependent focus formation, but were considerably more sensitive (Fig S2). We observed that Omicron infected the ACE2-expressing cells in a concentration dependent manner but did not infect the parental H1299 cells, indicating that ACE2 is required for Omicron entry (Fig. 1A). We then tested the ability of plasma from BNT162b2 vaccinated study participants to neutralize Omicron versus ancestral D614G virus in a live virus neutralization assay. We tested 14 plasma samples from 12 participants (Table S1), with 6 having no previous record of SARS-CoV-2 infection nor detectable nucleocapsid antibodies indicative of previous infection. For two of these participants, we used samples from two timepoints. The remaining 6 participants had a record of previous infection in the first SARS-CoV-2 infection wave in South Africa where infection was with ancestral D614G virus (Table S1). Geometric mean titer (GMT) FRNT50 (inverse of the plasma dilution required for 50% reduction in infection foci number) was 1321 for D614G. These samples therefore had very strong neutralization of D614G virus, consistent with sampling soon after vaccination. GMT FRNT50 for the same samples was 32 for Omicron, a 41-fold decline (Fig 1B). However, the escape was incomplete, with 5 of the participants, all previously infected, showing relatively high neutralization titers with Omicron. Beta variant escape from BNT162b2 in a live virus neutralization assay has been reported to be substantial (3) and our own data confirmed these results (4), with about 3-fold reduction in FRNT50. The results we present here with Omicron show much more extensive escape. However, escape was incomplete in participants with higher FRNT50 due to previous infection. Previous infection, followed by vaccination or booster is likely to increase the neutralization level and likely confer protection from severe disease in Omicron infection.

Antibody Response of BNT162b2 and CoronaVac Platforms in Recovered Individuals Previously Infected by COVID-19 against SARS-CoV-2 Wild Type and Delta Variant

Vaccinating recovered patients previously infected by COVID-19 with mRNA vaccines to boost their immune response against wild-type viruses (WT), we aimed to investigate whether vaccine platform and time of vaccination affect immunogenicity against the SARS-CoV-2 WT and Delta variant (DV). Convalescent patients infected by COVID-19 were recruited and received one booster dose of the BNT162b2 (PC-B) or CoronaVac (PC-C) vaccines, while SARS-CoV-2 naïve subjects received two doses of the BNT162b2 (CN-B) or CoronaVac (CN-C) vaccines. The neutralizing antibody in sera against the WT and DV was determined with live virus neutralization assay (vMN). The vMN geometric mean titre (GMT) against WT in recovered individuals previously infected by COVID-19 reduced significantly from 60.0 (95% confidence interval (CI), 46.5–77.4) to 33.9 (95% CI, 26.3–43.7) at 6 months post recovery. In the PC-B group, the BNT162b2 vaccine enhanced antibody response against WT and DV, with 22.3-fold and 20.4-fold increases, respectively. The PC-C group also showed 1.8-fold and 2.2-fold increases for WT and DV, respectively, after receiving the CoronaVac vaccine. There was a 10.6-fold increase in GMT in the CN-B group and a 1.3-fold increase in the CN-C group against DV after full vaccination. In both the PC-B and PC-C groups, there was no difference between GMT against WT and DV after vaccination. Subjects in the CN-B and CN-C groups showed inferior GMT against DV compared with GMT against WT after vaccination. In this study, one booster shot effectively enhanced the pre-existing neutralizing activity against WT and DV in recovered subjects.

Kinetics of Neutralizing Antibody Response Underscores Clinical COVID-19 Progression

Background. Neutralizing antibody (nAb) response is generated following infection or immunization and plays an important role in the protection against a broad of viral infections. The role of nAb during clinical progression of coronavirus disease 2019 (COVID-19) remains little known. Methods. 123 COVID-19 patients during hospitalization in Tongji Hospital were involved in this retrospective study. The patients were grouped based on the severity and outcome. The nAb responses of 194 serum samples were collected from these patients within an investigation period of 60 days after the onset of symptoms and detected by a pseudotyped virus neutralization assay. The detail data about onset time, disease severity and laboratory biomarkers, treatment, and clinical outcome of these participants were obtained from electronic medical records. The relationship of longitudinal nAb changes with each clinical data was further assessed. Results. The nAb response in COVID-19 patients evidently experienced three consecutive stages, namely, rising, stationary, and declining periods. Patients with different severity and outcome showed differential dynamics of the nAb response over the course of disease. During the stationary phase (from 20 to 40 days after symptoms onset), all patients evolved nAb responses. In particular, high levels of nAb were elicited in severe and critical patients and older patients (≥60 years old). More importantly, critical but deceased COVID-19 patients showed high levels of several proinflammation cytokines, such as IL-2R, IL-8, and IL-6, and anti-inflammatory cytokine IL-10 in vivo, which resulted in lymphopenia, multiple organ failure, and the rapidly decreased nAb response. Conclusion. Our results indicate that nAb plays a crucial role in preventing the progression and deterioration of COVID-19, which has important implications for improving clinical management and developing effective interventions.

Similar antibody responses against SARS-CoV-2 in HIV uninfected and infected individuals on antiretroviral therapy during the first South African infection wave

Background There is limited understanding of SARS-CoV-2 pathogenesis in African populations with a high burden of infectious disease comorbidities such as HIV. The kinetics, magnitude and duration of virus-specific antibodies and the underlying B cell responses in people living with HIV (PLWH) in sub-Saharan Africa have not been fully characterized. Methods We longitudinally followed SARS-CoV-2 infected individuals in Durban, KwaZulu-Natal, South Africa and characterized SARS-CoV-2 receptor binding domain-specific IgM, IgG and IgA antibodies weekly for a month, and then at 3 months post diagnosis. 7/30 (41.7%) were PLWH, 83% (25/30) of which were on ART and with full HIV suppression. Potency of convalescent plasma neutralization was determined using a live virus neutralization assay and antibody secreting cell population frequencies were determined by flow cytometry. Results Similar seroconversion rates, time to peak antibody titer, peak magnitude and durability of anti-SARS-CoV-2 IgM, IgG, IgA, were observed in HIV uninfected and PLWH with complete HIV suppression on ART. In addition, similar neutralization potency against an isolate of SARS-CoV-2, circulating at the time of sampling in the first wave of SARS-CoV-2 infections in South Africa was observed in both groups. Loss of IgA was significantly associated with age (p=0.023) and a previous diagnosis of TB (p=0.018). Conclusions Similar antibody response kinetics and neutralization potency in HIV negative and PLWH on stable ART in an African setting suggests that COVID-19 natural infections may confer comparable antibody immunity in these groups. This provides hope that COVID-19 vaccines will be effective in PLWH on stable ART.

Serological Evidence of Phleboviruses in Domestic Animals on the Pre-Apennine Hills (Northern Italy)

Phleboviruses are arboviruses transmitted by sand flies, mosquitoes and ticks. Some sand fly-borne phleboviruses cause illnesses in humans, such as the summer fevers caused by the Sicilian and Naples viruses or meningitis caused by the Toscana virus. Indeed, traces of several phleboviral infections have been serologically detected in domestic animals, but their potential pathogenic role in vertebrates other than humans is still unclear, as is the role of vertebrates as potential reservoirs of these viruses. In this study, we report the results of a serological survey performed on domestic animals sampled in Northern Italy, against four phleboviruses isolated from sand flies in the same area. The sera of 23 dogs, 165 sheep and 23 goats were tested with a virus neutralization assay for Toscana virus, Fermo virus, Ponticelli I virus and Ponticelli III virus. Neutralizing antibodies against one or more phleboviruses were detected in four out of 23 dogs, 31 out of 165 sheep and 12 out of 23 goats. This study shows preliminary evidence for the distribution pattern of phleboviral infections in different animal species, highlighting the potential infection of the Toscana virus in dogs and the Fermo virus in goats.

Broadly cross-reactive human antibodies that inhibit genogroup I and II noroviruses

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.

Infectivity and immune escape of the new SARS-CoV-2 variant of interest Lambda

Background: The newly described SARS-CoV-2 lineage C.37 was recently classified as a variant of interest by the WHO (Lambda variant) based on its high circulation rates in South American countries and the presence of critical mutations in the spike protein. The impact of such mutations in infectivity and immune escape from neutralizing antibodies are entirely unknown. Methods: We performed a pseudotyped virus neutralization assay and determined the impact of the Lambda variant on infectivity and immune escape using plasma samples from healthcare workers (HCW) from two centers in Santiago, Chile who received the two-doses scheme of the inactivated virus vaccine CoronaVac. Results: We observed an increased infectivity mediated by the Lambda spike protein that was even higher than that of the D614G (lineage B) or the Alpha and Gamma variants. Compared to the Wild type (lineage A), neutralization was decreased by 3.05-fold for the Lambda variant while it was 2.33-fold for the Gamma variant and 2.03-fold for the Alpha variant. Conclusions: Our results indicate that mutations present in the spike protein of the Lambda variant of interest confer increased infectivity and immune escape from neutralizing antibodies elicited by CoronaVac. These data reinforce the idea that massive vaccination campaigns in countries with high SARS-CoV-2 circulation must be accompanied by strict genomic surveillance allowing the identification of new isolates carrying spike mutations and immunology studies aimed to determine the impact of these mutations in immune escape and vaccines breakthrough.