Different Neutralization Profiles After Primary SARS-CoV-2 Omicron BA.1 and BA.2 Infections.

Background and Methods: The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron (B.1.1.529) variant is the antigenically most distinct variant to date. As the heavily mutated spike protein enables neutralization escape, we studied serum-neutralizing activities of naïve and vaccinated individuals after Omicron BA.1 or BA.2 sub-lineage infections in live virus neutralization tests with Omicron BA.1, Omicron BA.2, wildtype (WT, B1.1), and Delta (B.1.617.2) strains. Serum samples obtained after WT infections and three-dose mRNA vaccinations with and without prior infection were included as controls. Results: Primary BA.1 infections yielded reduced neutralizing antibody levels against WT, Delta, and Omicron BA.2, while samples from BA.2-infected individuals showed almost no cross-neutralization against the other variants. Serum neutralization of Omicron BA.1 and BA.2 variants was detectable after three-dose mRNA vaccinations, but with reduced titers. Vaccination-breakthrough infections with either Omicron BA.1 or BA.2, however, generated equal cross-neutralizing antibody levels against all SARS-CoV-2 variants tested. Conclusions: Our study demonstrates that although Omicron variants are able to enhance cross-neutralizing antibody levels in pre-immune individuals, primary infections with BA.1 or BA.2 induced mostly variant-specific neutralizing antibodies, emphasizing the differently shaped humoral immunity induced by the two Omicron variants. These data thus contribute substantially to the understanding of antibody responses induced by primary Omicron infections or multiple exposures to different SARS-CoV-2 variants and are of particular importance for developing vaccination strategies in the light of future emerging variants.

Heterogeneous SARS-CoV-2-Neutralizing Activities After Infection and Vaccination.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with different resistance levels to existing immunity have recently emerged. Antibodies that recognize the SARS-CoV-2 spike (S) protein and exhibit neutralizing activities are considered the best correlate of protection and an understanding of humoral immunity is crucial for controlling the pandemic. We thus analyzed such antibodies in individuals recovered from infection in 2020 as well as vaccinees after two doses of an mRNA vaccine. Methods: Neutralizing antibody responses against three SARS-CoV-2 variants (D614G, VOCs Beta and Delta) were determined in serum samples from 54 infected individuals (24 non-hospitalized, 30 hospitalized) and 34 vaccinees shortly after symptom onset or second vaccination, respectively, as well as six months later. In addition, the effect of the S sequence of the infecting strain on neutralization was studied. Results: Non-hospitalized patients had the lowest neutralization titers against all variants, while those of hospitalized patients equaled or exceeded those of vaccinees. Neutralizing activity was lower against the two VOCs and declined significantly in all cohorts after six months. This decrease was more pronounced in hospitalized and vaccinated individuals than in non-hospitalized patients. Of note, the specific neutralizing activity (NT titer/ELISA value ratio) was higher in the infected cohorts than in vaccinees and did not differ between non-hospitalized and hospitalized patients. Patients infected with viral strains carrying mutations in the N-terminal domain of the spike protein were impaired in Beta VOC neutralization. Conclusions: Specific neutralizing activities were higher in infected than in vaccinated individuals, and no difference in the quality of these antibodies was observed between hospitalized and non-hospitalized patients, despite significantly lower titers in the latter group. Additionally, antibody responses of infected individuals showed greater heterogeneity than those of vaccinees, which was associated with mutations in the spike protein of the infecting strain. Overall, our findings yielded novel insights into SARS-CoV-2-specific neutralizing antibodies, evolving differently after virus infection and COVID-19 vaccination, which is an important issue to consider in ongoing vaccine strategy improvements.

Invaplex functions as an intranasal adjuvant for subunit and DNA vaccines co-delivered in the nasal cavity of nonhuman primates.

Development of intranasal vaccines for HIV-1 and other mucosal pathogens has been hampered by the lack of adjuvants that can be given safely to humans. We have found that an intranasal Shigella vaccine (Invaplex) which is well tolerated in humans can also function as an adjuvant for intranasal protein and DNA vaccines in mice. To determine whether Invaplex could potentially adjuvant similar vaccines in humans, we simultaneously administered a simian immunodeficiency virus (SIV) envelope (Env) protein and DNA encoding simian-human immunodeficiency virus (SHIV) with or without Invaplex in the nasal cavity of female rhesus macaques. Animals were intranasally boosted with adenoviral vectors expressing SIV env or gag,pol to evaluate memory responses. Anti-SIV antibodies in sera and nasal, genital tract and rectal secretions were quantitated by ELISA. Intracellular cytokine staining was used to measure Th1-type T cells in blood. Macaques given DNA/protein immunizations with 0.5 mg Invaplex developed greater serum IgG, nasal IgA and cervicovaginal IgA responses to SIV Env and SHIV Gag,Pol proteins when compared to non-adjuvanted controls. Rectal IgA responses to Env were only briefly elevated and not observed to Gag,Pol. Invaplex increased frequencies of IFNγ-producing CD4 and CD8 T cells to the Env protein, but not T cell responses induced by the DNA. Ad-SIV boosting increased Env-specific polyfunctional T cells and Env- and Gag,Pol-specific antibodies in serum and all secretions. The data suggest that Invaplex could be highly effective as an adjuvant for intranasal protein vaccines in humans, especially those intended to prevent infections in the genital or respiratory tract.

Early host responses of seasonal and pandemic influenza A viruses in primary well-differentiated human lung epithelial cells.

Replication, cell tropism and the magnitude of the host's antiviral immune response each contribute to the resulting pathogenicity of influenza A viruses (IAV) in humans. In contrast to seasonal IAV in human cases, the 2009 H1N1 pandemic IAV (H1N1pdm) shows a greater tropism for infection of the lung similar to H5N1. We hypothesized that host responses during infection of well-differentiated, primary human bronchial epithelial cells (wd-NHBE) may differ between seasonal (H1N1 A/BN/59/07) and H1N1pdm isolates from a fatal (A/KY/180/10) and nonfatal (A/KY/136/09) case. For each virus, the level of infectious virus and host response to infection (gene expression and apical/basal cytokine/chemokine profiles) were measured in wd-NHBE at 8, 24, 36, 48 and 72 hours post-infection (hpi). At 24 and 36 hpi, KY/180 showed a significant, ten-fold higher titer as compared to the other two isolates. Apical cytokine/chemokine levels of IL-6, IL-8 and GRO were similar in wd-NHBE cells infected by each of these viruses. At 24 and 36 hpi, NHBE cells had greater levels of pro-inflammatory cytokines including IFN-α, CCL2, TNF-α, and CCL5, when infected by pandemic viruses as compared with seasonal. Polarization of IL-6 in wd-NHBE cells was greatest at 36 hpi for all isolates. Differential polarized secretion was suggested for CCL5 across isolates. Despite differences in viral titer across isolates, no significant differences were observed in KY/180 and KY/136 gene expression intensity profiles. Microarray profiles of wd-NHBE cells diverged at 36 hpi with 1647 genes commonly shared by wd-NHBE cells infected by pandemic, but not seasonal isolates. Significant differences were observed in cytokine signaling, apoptosis, and cytoskeletal arrangement pathways. Our studies revealed differences in temporal dynamics and basal levels of cytokine/chemokine responses of wd-NHBE cells infected with each isolate; however, wd-NHBE cell gene intensity profiles were not significantly different between the two pandemic isolates suggesting post-transcriptional or later differences in viral-host interactions.

Phenotypic differences in virulence and immune response in closely related clinical isolates of influenza A 2009 H1N1 pandemic viruses in mice.

To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm) strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients hospitalized during 2009-2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNγ, MIG, and IL-10 responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice. Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung, bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages and this may be a key determinant in tipping the response toward a more severe disease progression.

Genetic immunization in the lung induces potent local and systemic immune responses.

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge.

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.