Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Difference in Strain Pathogenicity of Septicemic Yersinia pestis Infection in a TLR2-/- Mouse Model.

Yersinia pestis is the causative agent of bubonic, pneumonic, and septicemic plague. We demonstrate that Toll-like receptor 2-deficient (TLR2-/-) mice are resistant to septicemic infection by the KIM5 strain of Y. pestis but not to infection by the CO92 Δpgm strain. This resistance is dependent on TLR2, the route of infection, and the isoform of YopJ. Elevated bacterial burdens were found in the spleens of CO92 Δpgm-infected animals by 24 h postinfection and in the livers by 4 days. The YopJ isoform present contributed directly to cytotoxicity and inflammatory cytokine production of bone marrow-derived macrophages from TLR2-/- mice. Immune cell trafficking is altered in CO92 Δpgm infections, with an increased neutrophil infiltration to the spleen 5 days postinfection. Immune cell infiltration to the liver was greater and earlier in KIM5-infected TLR2-/- mice. The functionality of the immune cells was assessed by the ability to develop reactive oxygen and nitrogen species. Our data suggest an inhibition of granulocytes in forming these species in CO92 Δpgm-infected TLR2-/- mice. These findings suggest that resistance to KIM5 in TLR2-/- mice is dependent on early immune cell trafficking and functionality.

Cloning and functional testing of rhesus macaque (Macaca mulatta) IL-9 and IL-33.

BACKGROUND: IL-9 and IL-33 can profoundly influence immune responses. As a necessary first step toward defining their impact in the rhesus macaque model, we confirmed their endogenous expression and sequence identity and generated expression vectors for the recombinant expression of rhesus IL-9 and IL-33. METHODS: RT-PCR and Sanger sequencing was used to define the expression and sequences for rhesus IL-9 and IL-33. The resulting recombinant cytokines were tested by ELISA and proliferation assays. RESULTS: Full-length rhesus IL-9 and the mature form of rhesus IL-33 share 78% and 73% nucleotide similarity, respectively, with humans. Both cytokines are expressed in lymphocytes, with IL-9 expression also evident in CD4+ T cells. Recombinantly expressed rhesus IL-9 and IL-33 were each biologically active in vitro, including enhancing the proliferation of a rhesus B cell line. CONCLUSIONS: The recombinant rhesus IL-9 and IL-33 constructs produce biologically active cytokines that can act upon rhesus B cells.

Injection Drug Use Alters Plasma Regulation of the B Cell Response.

The opioid epidemic continues to be a major public health issue that includes millions of people who inject drugs (PWID). PWID have increased incidence of serious infections, including HIV as well as metabolic and inflammatory sequelae. We sought to discern the extent of systemic alterations in humoral immunity associated with injection drug use, including alterations in the plasma proteome and its regulation of B cell responsiveness. Comprehensive plasma proteomics analysis of HIV negative/hepatitis C negative individuals with a history of recent injection heroin use was performed using mass spectrometry and ELISA. The effects of plasma from PWID and healthy controls on the in vitro proliferation and transcriptional profile of B cell responses to stimulation were determined by flow cytometry and RNA-Seq. The plasma proteome of PWID was distinct from healthy control individuals, with numerous immune-related analytes significantly altered in PWID, including complement (C3, C5, C9), immunoglobulin (IgD, IgM, kappa light chain), and other inflammatory mediators (CXCL4, LPS binding protein, C-reactive protein). The plasma of PWID suppressed the in vitro proliferation of B cells. Transcriptome analysis indicated that PWID plasma treatment increased B cell receptor and CD40 signaling and shifted B cell differentiation from plasma cell-like toward germinal center B cell-like transcriptional profiles. These results indicate that the systemic inflammatory milieu is substantially altered in PWID and may impact their B cell responses.

Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants.

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1Å resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Duration of post-COVID-19 symptoms is associated with sustained SARS-CoV-2-specific immune responses.

A subset of COVID-19 patients exhibit post-acute sequelae of COVID-19 (PASC), but little is known about the immune signatures associated with these syndromes. We investigated longitudinal peripheral blood samples in 50 individuals with previously confirmed SARS-CoV-2 infection, including 20 who experienced prolonged duration of COVID-19 symptoms (lasting more than 30 days; median = 74 days) compared with 30 who had symptom resolution within 20 days. Individuals with prolonged symptom duration maintained antigen-specific T cell response magnitudes to SARS-CoV-2 spike protein in CD4+ and circulating T follicular helper cell populations during late convalescence, while those without persistent symptoms demonstrated an expected decline. The prolonged group also displayed increased IgG avidity to SARS-CoV-2 spike protein. Significant correlations between symptom duration and both SARS-CoV-2-specific T cells and antibodies were observed. Activation and exhaustion markers were evaluated in multiple immune cell types, revealing few phenotypic differences between prolonged and recovered groups, suggesting that prolonged symptom duration is not due to persistent systemic inflammation. These findings demonstrate that SARS-CoV-2-specific immune responses are maintained in patients suffering from prolonged post-COVID-19 symptom duration in contrast to those with resolved symptoms and may suggest the persistence of viral antigens as an underlying etiology.

CD4+ T Cells Are Dispensable for Induction of Broad Heterologous HIV Neutralizing Antibodies in Rhesus Macaques.

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies.

Sustained cellular immune dysregulation in individuals recovering from SARS-CoV-2 infection.

SARS-CoV-2 causes a wide spectrum of clinical manifestations and significant mortality. Studies investigating underlying immune characteristics are needed to understand disease pathogenesis and inform vaccine design. In this study, we examined immune cell subsets in hospitalized and nonhospitalized individuals. In hospitalized patients, many adaptive and innate immune cells were decreased in frequency compared with those of healthy and convalescent individuals, with the exception of an increase in B lymphocytes. Our findings show increased frequencies of T cell activation markers (CD69, OX40, HLA-DR, and CD154) in hospitalized patients, with other T cell activation/exhaustion markers (PD-L1 and TIGIT) remaining elevated in hospitalized and nonhospitalized individuals. B cells had a similar pattern of activation/exhaustion, with increased frequency of CD69 and CD95 during hospitalization followed by an increase in PD1 frequencies in nonhospitalized individuals. Interestingly, many of these changes were found to increase over time in nonhospitalized longitudinal samples, suggesting a prolonged period of immune dysregulation after SARS-CoV-2 infection. Changes in T cell activation/exhaustion in nonhospitalized patients were found to positively correlate with age. Severely infected individuals had increased expression of activation and exhaustion markers. These data suggest a prolonged period of immune dysregulation after SARS-CoV-2 infection, highlighting the need for additional studies investigating immune dysregulation in convalescent individuals.

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters.

SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

IL-33 enhances the kinetics and quality of the antibody response to a DNA and protein-based HIV-1 Env vaccine.

Induction of a sustained and broad antibody (Ab) response is a major goal in developing a protective HIV-1 vaccine. DNA priming alone shows reduced levels of immunogenicity; however, when combined with protein boosting is an attractive vaccination strategy for induction of humoral responses. Using the VC10014 DNA and protein-based vaccine consisting of HIV-1 envelope (Env) gp160 plasmids and trimeric gp140 proteins derived from an HIV-1 clade B infected subject who developed broadly neutralizing serum Abs, and which has been previously demonstrated to induce Tier 2 heterologous neutralizing Abs in rhesus macaques, we evaluated whether MPLA and IL-33 when administered during the DNA priming phase enhances the humoral response in mice. The addition of IL-33 during the gp160 DNA priming phase resulted in high titer gp120-specific plasma IgG after the first immunization. The IL-33 treated mice had higher plasma IgG Ab avidity, breadth, and durability after DNA and protein co-immunization with alum adjuvant as compared to MPLA and alum only treated mice. IL-33 was also associated with a significant IgM Env-specific response and expansion of peritoneal and splenic B-1b B cells. These results indicate that DNA priming in the presence of exogenous IL-33 qualitatively alters the HIV-1 Env-specific humoral response, improving the kinetics and breadth of potentially protective Ab.