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.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection.

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Molecular cloning of IgZ heavy chain isotype in Catla catla and comparative expression profile of IgZ and IgM following pathogenic infection.

Immunoglobulins serve as a crucial arm of the adaptive immune system against detrimental pathogenic threats in teleosts. However, whether the novel Ig isotype IgZ is present in the Indian major carp, Catla catla, has not yet been elucidated. The present study reports the presence of IgZ ortholog in C. catla (CcIgZ) and further demonstrates its comparative tissue specific expression with IgM (CcIgM) in response to bacterial and parasitic stimulation. The putative 139 amino acid sequence of IgZ heavy chain cDNA of C. catla showed homology with IgZ constant domains of other teleosts. Phylogenetic analysis of the predicted IgZ transcript sequence clustered with previously identified IgZ heavy chain sequences of Cyprinidae family members. The inductive expression profiles of IgZ and IgM genes were evaluated in immunologically relevant tissues at 24, 48 and 72 hr post infection with Aeromonas hydrophila, Streptococcus uberis and Argulus sp. Both CcIgZ and CcIgM were expressed most strongly in the kidneys of healthy fish. Basal expression of CcIgM transcript was higher than that of CcIgZ in all the examined tissues. Stimulation with bacteria triggered significant increase of IgZ in the intestine (P < 0.001) and spleen (P < 0.01), whereas IgM was relatively up-regulated in blood (P < 0.001) after stimulation with each of the three pathogens assessed. The study is the first to report identification of IgZ in C. catla. Further, it provides insights into the differential expression profiles of IgZ and IgM isotypes against various pathogenic infection in C. catla, which may facilitate better prophylaxis again such infections.

Hypoxic stress: impact on the modulation of TLR2, TLR4, NOD1 and NOD2 receptor and their down-stream signalling genes expression in catla (Catla catla).

The damage-associated molecular patterns (DAMPs) released from the damaged tissue/cells are recently reported as endogenous ligands to activate toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors signaling pathways. In the aquatic environment, reduction in dissolved oxygen (DO) concentration causes hypoxic stress resulting in tissue damage and patho-biological changes in fish. We envisaged the critical role of TLR and NOD receptors in recognizing DAMPs as endogenous ligands during hypoxic stress in fish. Catla (Catla catla) fingerlings (avg. wt ~56 g) was exposed to hypoxic stress (DO: 1-3 mg/L) for 1 and 24 h. After the designated time course, total RNA was extracted from gill, liver, kidney and blood, and modulation of TLRs (TLR2 and TLR4), NOD (NOD1 and NOD2) receptors, MyD88 (myeloid differentiation primary response gene 88), RICK (receptor interacting serine-threonine protein kinase-2), interleukin (IL)-6, IL-8 and IL-10 gene expression were analyzed by quantitative reverse transcriptase PCR assay. Significant (p < 0.05) up-regulation of some DAMPs {high-mobility group box 1 and heat shock protein-70}, TLRs and NOD receptors genes expressions were observed in the hypoxic fish tissues as compared to the control. Further investigation revealed inductive expression of MyD88, RICK, IL-6, IL-8 and IL-10 genes in the TLRs and NODs activated tissues of the hypoxic fish. These data together may suggest the important role of TLRs and NOD receptors signaling pathway in sterile inflammation and pathobiology of fish in hypoxic stress, and warrant further study to investigate the role of TLR and NOD receptors in abiotic stress management in aquaculture.

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.

Molecular cloning and characterization of Toll-like receptor 3, and inductive expression analysis of type I IFN, Mx and pro-inflammatory cytokines in the Indian carp, rohu (Labeo rohita).

Toll-like receptors (TLRs) are one of the key components of innate or non-specific immunity. Among various types of TLRs, TLR3 recognizes dsRNA, the genetic material or replicative intermediate of many RNA viruses and triggers TIR-domain-containing adapter-inducing interferon-ß dependent signalling pathway to induce type I interferon (IFN) and pro-inflammatory cytokines. In this study, we cloned and characterized full-length TLR3 cDNA in rohu (Labeo rohita), that comprised of 2,619 bp nucleotides encoding a putative protein of 873 amino acid with the estimated molecular mass of 98.57 kDa. The constitutive expression of TLR3 gene was detected in all embryonic developmental stages and in various organs/tissues of rohu fingerlings. In vivo tissue specific modulation of TLR3, type I IFN, Mx (myxovirus-resistant protein) and pro-inflammatory cytokines (TNF-α and IL-1ß) gene expression were analysed by quantitative real-time PCR following intravenous injection of polyinosinic-polycytidylic acid (poly I:C), a synthetic analogue of viral dsRNA. A significant relationship of TLR3 induction, and type I IFN, Mx, IL-1ß and TNF-α gene expression were observed in majority of the treated fish tissues, as compared to their control. Together, these data highlight the important role of TLR3 in recognizing dsRNA, and in augmenting the innate immunity in fish in response to viral infections.

Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

Fc-Effector-Independent in vivo Activity of a Potent Influenza B Neuraminidase Broadly Neutralizing Antibody.

Influenza B virus (IBV) contributes to substantial influenza-mediated morbidity and mortality, particularly among children. Similar to influenza A viruses (IAV), the hemagglutinin (HA) and neuraminidase (NA) of IBV undergo antigenic drift, necessitating regular reformulation of seasonal influenza vaccines. NA inhibitors, such as oseltamivir, have reduced activity and clinical efficacy against IBV, while M2 channel inhibitors are only effective against IAV, highlighting the need for improved vaccine and therapeutics for the treatment of seasonal IBV infections. We have previously described a potent human monoclonal antibody (hMAb), 1092D4, that is specific for IBV NA and neutralizes a broad range of IBVs. The anti-viral activity of MAbs can include direct mechanisms such as through neutralization and/or Fc-mediated effector functions that are dependent on accessory cells expressing Fc receptors and that could be impacted by potential host-dependent variability. To discern if the in vivo efficacy of 1092D4 was dependent on Fc-effector function, 1092D4 hMAb with reduced ability to bind to Fc receptors (1092D4-LALAPG) was generated and tested. 1092D4-LALAPG had comparable in vitro binding, neutralization, and inhibition of NA activity to 1092D4. 1092D4-LALAPG was effective at protecting against a lethal challenge of IBV in mice. These results suggest that hMAb 1092D4 in vivo activity is minimally dependent on Fc-effector functions, a characteristic that may extend to other hMAbs that have potent NA inhibition activity.

Mixed Origins: HIV gp120-Specific Memory Develops from Pre-Existing Memory and Naive B Cells Following Vaccination in Humans.

The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env, suggesting that the initial responding B cell population not only exclusively comprises a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing, focused on immunoglobulin G (IgG), IgA, and IgM, VH-lineage tracking, we identified four of these mAb lineages in pre-immune peripheral blood. We also looked through the ∼7 month postvaccination bone marrow, and interestingly, several of these lineages that were found in prevaccination blood were still persistent in the postvaccination bone marrow, including the CD138+ long-lived plasma cell compartment. The majority of the pre-immune lineage members included IgM, however, IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells. ClinicalTrials.gov NCT02207920.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Molecular cloning and characterization of nucleotide binding and oligomerization domain-1 (NOD1) receptor in the Indian Major Carp, rohu (Labeo rohita), and analysis of its inductive expression and down-stream signalling molecules following ligands exposure and Gram-negative bacterial infections.

Nucleotide binding and oligomerization domain-1 (NOD1) is a cytoplasmic pattern recognition receptor (PRR), and is a member of the NOD-like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products, and plays a key role in inducing innate immunity. In this report, NOD1 gene was cloned and characterized in rohu (Labeo rohita), a fish species of highest commercial importance in the Indian subcontinent. The full-length rohu NOD1 (rNOD1) cDNA comprised of 3168 bp with a single open reading frame (ORF) of 2814 bp, encoding a polypeptide of 937 amino acids (aa) with an estimated molecular mass of 106.13 kDa. Structurally, it comprised of one caspase recruitment domain (CARD) at N-terminal, seven leucine rich repeat (LRR) regions at C-terminal and one NACHT domain in between N and C-terminals. Phylogenetically, rNOD1 was closely related to grass carp NOD1 (gcNOD1), and exhibited significant similarity (95.8%) and identity (91.0%) in their amino acids. Ontogenic expression analysis of rNOD1 and its associated down-stream signaling molecule RICK (receptor interacting serine­threonine kinase) by quantitative real-time PCR (qRT-PCR) revealed their constitutive expression in all embryonic developmental stages. Basal expression analysis of rNOD1 showed its wide range of expression in all examined tissues, highest was in spleen and the lowest was in blood. Inductive expression of rNOD1 was observed following LPS and poly I:C exposure, and Aeromonas hydrophila, Edwardsiella tarda and Shigella flexneri infections. Expression of RICK in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with the inductive expression of rNOD1. Together, these findings highlighted the important role of NOD1 in fish in response to pathogenic invasion.

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita).

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.

Induction of toll-like receptor (TLR) 2, and MyD88-dependent TLR- signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala).

Toll-like receptor 2 (TLR2) is a member of TLR family. It recognizes a wide range of bacteria and their products, and is involved in inducing innate immune responses. In this article, we reported inductive expression of TLR2 and myeloid differentiation primary response gene 88 (MyD88)-dependent signaling in the Indian major carp, mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) genes by quantitative real-time PCR (qRT-PCR) revealed constitutive expression of these genes in all embryonic developmental stages, indicating their involvement in embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by qRT-PCR showed their wide distribution in various organs and tissues. Highest expression of TLR2 was in gill, MyD88 in liver and TRAF6 was in kidney. Inductive expression of TLR2, MyD88 and TRAF6 genes were observed following peptidoglycan (PGN)-treatment, and Streptococcus uberis and Aeromonas hydrophila infections. Expression of interleukin (IL)-8 and TNF-α in various organs were significantly enhanced by PGN-treatment and bacterial infections, and were closely associated with TLR2 induction. These findings together highlighted the contribution of TLR2 in augmenting innate immunity in fish, and indicated it's important role in immune surveillance of various organs during pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish, and will be helpful in developing preventive measures against infectious diseases in fish.

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.

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.

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.

Persistence of HIV-1 Env-Specific Plasmablast Lineages in Plasma Cells after Vaccination in Humans.

Induction of persistent HIV-1 Envelope (Env) specific antibody (Ab) is a primary goal of HIV vaccine strategies; however, it is unclear whether HIV Env immunization in humans induces bone marrow plasma cells, the presumed source of long-lived systemic Ab. To define the features of Env-specific plasma cells after vaccination, samples were obtained from HVTN 105, a phase I trial testing the same gp120 protein immunogen, AIDSVAX B/E, used in RV144, along with a DNA immunogen in various prime and boost strategies. Boosting regimens that included AIDSVAX B/E induced robust peripheral blood plasmablast responses. The Env-specific immunoglobulin repertoire of the plasmablasts is dominated by VH1 gene usage and targeting of the V3 region. Numerous plasmablast-derived immunoglobulin lineages persisted in the bone marrow >8 months after immunization, including in the CD138+ long-lived plasma cell compartment. These findings identify a cellular linkage for the development of sustained Env-specific Abs following vaccination in humans.

Toll like receptor induces Ig synthesis in Catla catla by activating MAPK and NF-κB signalling.

The molecular crosstalk of proximal innate immune receptor signaling mediated by Toll-like receptors (TLRs) is crucial in generating an adaptive immune response. The extracellular-signal regulated kinases (ERK) participate in propagating intracellular signals initiated by stimulated TLRs to transcription factors eliciting cytokine release. Although ERK signaling has been extensively studied in mammalian counterparts, very little is known about its existence in carps and its role in augmentation of immunoglobulin (Ig) synthesis. Therefore, to gain insights into the efficacy of MAP kinase cascade in orchestrating fish antigen receptor generation, Catla catla fingerlings were induced with various TLR agonists or pathogen associated molecular patterns (PAMPs). Analysis of upstream signaling events revealed that PAMPs stimulated the tissues leading to a significant upregulation (P < 0.001, One-way ANOVA) of different TLRs (TLR2, TLR3, TLR4 and TLR5) followed by activation of MyD88 dependent and independent pathway. Activation of ERK and NF-κB mediated cytokine production consequently triggered the enhanced expression of IgZ and IgM as was evident by qRT-PCR analysis, flow cytometry, immunoblotting and ELISA. Pretreatment with ERK inhibitor (UO126) antagonized PAMPs mediated TLR stimulation, leading to sequential downregulation of MyD88/NF-κB/cytokines via interrupting ERK/NF-κB signaling axis. Together these results demonstrate that TLR stimulation triggers IgZ and IgM production via activation of ERK and NF-κB in C. catla indicating that NF-κB mediated cytokine production and ERK1/2 signaling is not only functional in fish, but may be crucial for generation of Ig repertoire in lower vertebrates.

Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells.

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results represent the first demonstration that IIV-induced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development.IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.