Potently neutralizing and protective human antibodies against SARS-CoV-2

The COVID-19 pandemic is a major threat to global health1 for which there are limited medical countermeasures2,3. Moreover, we currently lack a thorough understanding of mechanisms of humoral immunity4. From a larger panel of human monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein5, we identified several that exhibited potent neutralizing activity and fully blocked the receptor-binding domain of S (SRBD) from interacting with human ACE2 (hACE2). Competition-binding, structural, and functional studies allowed clustering of the mAbs into classes recognizing distinct epitopes on the SRBD as well as distinct conformational states of the S trimer. Potent neutralizing mAbs recognizing non-overlapping sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two mouse models of SARS-CoV-2 infection, passive transfer of either COV2-2196 or COV2-2130 alone or a combination of both mAbs protected mice from weight loss and reduced viral burden and inflammation in the lung. In addition, passive transfer of each of two of the most potently ACE2 blocking mAbs (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutics

Inferior immunogenicity and efficacy of respiratory syncytial virus fusion protein-based subunit vaccine candidates in aged versus young mice.

Respiratory syncytial virus (RSV) is recognized as an important cause of lower and upper respiratory tract infections in older adults, and a successful vaccine would substantially lower morbidity and mortality in this age group. Recently, two vaccine candidates based on soluble purified glycoprotein F (RSV F), either alone or adjuvanted with glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE), failed to reach their primary endpoints in clinical efficacy studies, despite demonstrating the desired immunogenicity profile and efficacy in young rodent models. Here, one of the RSV F vaccine candidates (post-fusion conformation, RSV post-F), and a stabilized pre-fusion form of RSV F (RSV pre-F, DS-Cav1) were evaluated in aged BALB/c mice. Humoral and cellular immunogenicity elicited after immunization of naïve, aged mice was generally lower compared to young animals. In aged mice, RSV post-F vaccination without adjuvant poorly protected the respiratory tract from virus replication, and addition of GLA-SE only improved protection in the lungs, but not in nasal turbinates. RSV pre-F induced higher neutralizing antibody titers compared to RSV post-F (as previously reported) but interestingly, RSV F-specific CD8 T cell responses were lower compared to RSV post-F responses regardless of age. The vaccines were also tested in RSV seropositive aged mice, in which both antigen forms similarly boosted neutralizing antibody titers, although GLA-SE addition boosted neutralizing activity only in RSV pre-F immunized animals. Cell-mediated immune responses in the aged mice were only slightly boosted and well below levels induced in seronegative young mice. Taken together, the findings suggest that the vaccine candidates were not able to induce a strong anti-RSV immune response in recipient mice with an aged immune system, in agreement with recent human clinical trial results. Therefore, the aged mouse model could be a useful tool to evaluate improved vaccine candidates, targeted to prevent RSV disease in older adults

Immunogenicity and protection from viral challenge of pre-RSV + GLA-SE in young, seronegative mice.

<p>Young (7 weeks old) BALB/c mice (n = 5 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F pre- and post-conformation at 0.3 μg +/- GLA-SE (2.5 μg/2%) adjuvant. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. (A) Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. (B) 4 days post-challenge (day 39), splenocytes were isolated and stimulated for 24 h with RSV F specific CD4 T-cell and CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. (C) Lung and (D) nasal turbinates viral loads were measured by plaque assay. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Lung T cell profile after RSV challenge in young and aged seronegative mice immunized with post-RSV F formulations.

<p>Young (7 weeks old) and aged (18 months old) BALB/c mice (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 with buffer alone, post-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. 4 days post-challenge (day 39), lung cells were isolated and stimulated with RSV F peptide pool to evaluate intracellular cytokine expression by flow cytometry. Cells were surface stained for CD3 and CD8, intracellularly stained for IFNγ, TNFα, and IL-2, and analyzed on an LSR II for the frequency of responding (A) CD4+ and (B) CD8+ T cells. (C) The percentage of F85-93-pentamer+ CD8 T cells was determined by flow cytometry. (D) The percentage of lung eosinophils was determined by flow cytometry. For (C) and (D), the mean of individual values +/- SD is shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Immunogenicity of post-F and pre-F in aged seronegative mice.

<p>Aged (18 months old) BALB/c mice (n = 6 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F (pre- or post- conformation) at the indicated doses +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. 4 days post-RSV A2 challenge, splenocytes were isolated and stimulated for 24 h with peptides representing <b>(B)</b> RSV F specific CD4 T cell and <b>(C)</b> CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Protection from RSV A2 challenge in aged seronegative mice, immunized with pre- and post-RSV F.

<p>Aged (18 months old) BALB/c mice (n = 6 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F (pre- or post- conformation) at the indicated doses +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. 4 days post-RSV A2 challenge, <b>(A)</b> lung and <b>(B)</b> nasal turbinates viral loads were measured by plaque assay. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Lung T cell profile of aged seropositive mice following immunization with different RSV F formulations and post challenge with RSV A2.

<p>12 months old mice were i.n. immunized with RSV A2 (10⁶ PFU) at day 0 and RSV B (10⁵ PFU) at day 60. Aged seropositive mice were then immunized i.m. at day 176 (18 months old) with buffer alone, post-F or pre-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. At day 190, aged seropositive mice were challenged with RSV A2 (10⁶ PFU). 4 days later, lung cells were isolated and stimulated with RSV F peptide pool. Cells were then surface stained for CD3 and CD8, intracellularly stained for IFNγ, TNFα, and IL-2, and analyzed on an LSR II for the frequency of responding <b>(A)</b> CD4+ and <b>(B)</b> CD8+ T cells.</p

NAb induction of pre-F and post-F formulations in aged seropositive mice.

<p>12 months old mice were i.n. immunized with RSV A2 (10⁶ PFU) at day 0 and RSV B (10⁵ PFU) at day 60. Aged seropositive mice were then immunized i.m. at day 176 (18 months old) with buffer alone, post-F or pre-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. <b>(A)</b> Bleeds were collected at day 50, 120, and 174 to assess the levels of RSV A2 neutralizing antibodies in seropositive animals prior to immunization (baseline). Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. <b>(B)</b> 14 days post immunization, RSV A2 neutralization titers were evaluated. Group means ± SD of individual mice are shown. For statistical analyses, multiplicity adjusted ANOVA test was performed against baseline as a control group, followed by pairwise testing of post-F versus pre-F, * for P<0.05.</p

Immunogenicity of post- RSV F formulations in young and old seronegative mice.

<p>Young (7 weeks old) and aged (18 months old) BALB/c mice (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 with buffer alone, post-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. (A) Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. At sacrifice (day 39), (B) RSV F specific anti-IgG1 and anti-IgG2a were measured by ELISA, and splenocytes were isolated and stimulated for 24 h with peptides representing (C) RSV F specific CD4 T-cell and (D) CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p