Secreted phospholipase PLA2G2E contributes to regulation of T cell immune response against influenza virus infection.

The involvement of secreted phospholipase A2s in respiratory diseases, such as asthma and respiratory viral infections, is well-established. However, the specific role of secreted phospholipase A2 group IIE (PLA2G2E) during influenza virus infection remains unexplored. Here, we investigated the role of PLA2G2E during H1N1 influenza virus infection using a targeted mouse model lacking Pla2g2e gene (Pla2g2e-/-). Our findings demonstrated that Pla2g2e-/- mice had significantly lower survival rates and higher viral loads in lungs compared to wild-type mice following influenza virus infection. While Pla2g2e-/- mice displayed comparable innate and humoral immune responses to influenza virus challenge, the animals showed impaired influenza-specific cellular immunity and reduced T cell-mediated cytotoxicity. This indicates that PLA2G2E is involved in regulating specific T cell responses during influenza virus infection. Furthermore, transgenic mice expressing the human PLA2G2E gene exhibited resistance to influenza virus infection along with enhanced influenza-specific cellular immunity and T cell-mediated cytotoxicity. Pla2g2e deficiency resulted in perturbation of lipid mediators in the lung and T cells, potentially contributing to its impact on the anti-influenza immune response. Taken together, these findings suggest that targeting PLA2G2E could hold potential as a therapeutic strategy for managing influenza virus infections.IMPORTANCEThe influenza virus is a highly transmissible respiratory pathogen that continues to pose a significant public health concern. It effectively evades humoral immune protection conferred by vaccines and natural infection due to its continuous viral evolution through the genetic processes of antigenic drift and shift. Recognition of conserved non-mutable viral epitopes by T cells may provide broad immunity against influenza virus. In this study, we have demonstrated that phospholipase A2 group IIE (PLA2G2E) plays a crucial role in protecting against influenza virus infection through the regulation of T cell responses, while not affecting innate and humoral immune responses. Targeting PLA2G2E could therefore represent a potential therapeutic strategy for managing influenza virus infection.

Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Neutralizing monoclonal antibodies protect against human adenovirus type 55 infection in transgenic mice and tree shrews.

Re-emerging human adenovirus type 55 (HAdV55) has become a significant threat to public health due to its widespread circulation and the association with severe pneumonia, but an effective anti-HAdV55 agent remains unavailable. Herein, we report the generation of macaque-derived, human-like monoclonal antibodies (mAbs) protecting against HAdV55 infection with high potency. Using fluorophore-labelled HAdV55 virions as probes, we isolated specific memory B cells from rhesus macaques (Macaca mulatta) that were immunized twice with an experimental vaccine based on E1-, E3-deleted, replication-incompetent HAdV55. We cloned a total of 19 neutralizing mAbs, nine of which showed half-maximal inhibitory concentrations below 1.0 ng/ml. These mAbs recognized the hyper-variable-region (HVR) 1, 2, or 7 of viral hexon protein, or the fibre knob. In transgenic mice expressing human desmoglein-2, the major cellular receptor for HAdV55, a single intraperitoneal injection with hexon-targeting mAbs efficiently prevented HAdV55 infection, and mAb 29C12 showed protection at a dose as low as 0.004 mg/kg. Fibre-targeting mAb 28E8, however, showed protection only at a dose up to 12.5 mg/kg. In tree shrews that are permissive for HAdV55 infection and disease, mAb 29C12 effectively prevented HAdV55-caused pneumonia. Further analysis revealed that fibre-targeting mAbs blocked the attachment of HAdV55 to host cells, whereas hexon-targeting mAbs, regardless of their targeting HVRs, mainly functioned at post-attachment stage via inhibiting viral endosomal escape. Our results indicate that hexon-targeting mAbs have great anti-HAdV55 activities and warrant pre-clinical and clinical evaluation.

Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines.

IMPORTANCE: Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.

Effect of seasonal coronavirus immune imprinting on the immunogenicity of inactivated COVID-19 vaccination.

Background: Pre-existing cross-reactive immunity among different coronaviruses, also termed immune imprinting, may have a comprehensive impact on subsequent SARS-CoV-2 infection and COVID-19 vaccination effectiveness. Here, we aim to explore the interplay between pre-existing seasonal coronaviruses (sCoVs) antibodies and the humoral immunity induced by COVID-19 vaccination. Methods: We first collected serum samples from healthy donors prior to COVID-19 pandemic and individuals who had received COVID-19 vaccination post-pandemic in China, and the levels of IgG antibodies against sCoVs and SARS-CoV-2 were detected by ELISA. Wilcoxon rank sum test and chi-square test were used to compare the difference in magnitude and seropositivity rate between two groups. Then, we recruited a longitudinal cohort to collect serum samples before and after COVID-19 vaccination. The levels of IgG antibodies against SARS-CoV-2 S, S1, S2 and N antigen were monitored. Association between pre-existing sCoVs antibody and COVID-19 vaccination-induced antibodies were analyzed by Spearman rank correlation. Results: 96.0% samples (339/353) showed the presence of IgG antibodies against at least one subtype of sCoVs. 229E and OC43 exhibited the highest seroprevalence rates at 78.5% and 72.0%, respectively, followed by NL63 (60.9%) and HKU1 (52.4%). The levels of IgG antibodies against two ß coronaviruses (OC43 and HKU1) were significantly higher in these donors who had inoculated with COVID-19 vaccines compared to pre-pandemic healthy donors. However, we found that COVID-19 vaccine-induced antibody levels were not significant different between two groups with high levelor low level of pre-existing sCoVs antibody among the longitudinal cohort. Conclusion: We found a high prevalence of antibodies against sCoVs in Chinese population. The immune imprinting by sCoVs could be reactivated by COVID-19 vaccination, but it did not appear to be a major factor affecting the immunogenicity of COVID-19 vaccine. These findings will provide insights into understanding the impact of immune imprinting on subsequent multiple shots of COVID-19 vaccines.

A new vaccination regimen using adenovirus-vectored vaccine confers effective protection against African swine fever virus in swine.

African swine fever (ASF) is an acute and highly contagious lethal infectious disease in swine that severely threatens the global pig industry. At present, a safe and efficacious vaccine is urgently required to prevent and control the disease. In this study, we evaluated the safety and immunogenicity of replication-incompetent type-2 adenoviruses carrying African swine fever virus (ASFV) antigens, namely CP204L (p30), E183L (p54), EP402R (CD2v), B646L (p72), and B602L (p72 chaperone). A vaccine cocktail delivered by simultaneous intramuscular (IM) and intranasal (IN) administration robustly elicited both systemic and mucosal immune responses against AFSV in mice and swine and provided highly effective protection against the circulating ASFV strain in farmed pigs. This multi-antigen cocktail vaccine was well tolerated in the vaccinated animals. No significant interference among antigens was observed. The combined IM and IN vaccination using this adenovirus-vectored antigen cocktail vaccine warrants further evaluation for providing safe and effective protection against ASFV infection and transmission.

Entangling of Peptide Nanofibers Reduces the Invasiveness of SARS-CoV-2.

The viral spike (S) protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells, facilitating its entry and infection. Here, functionalized nanofibers targeting the S protein with peptide sequences of IRQFFKK, WVHFYHK and NSGGSVH, which are screened from a high-throughput one-bead one-compound screening strategy, are designed and prepared. The flexible nanofibers support multiple binding sites and efficiently entangle SARS-CoV-2, forming a nanofibrous network that blocks the interaction between the S protein of SARS-CoV-2 and the ACE2 on host cells, and efficiently reduce the invasiveness of SARS-CoV-2. In summary, nanofibers entangling represents a smart nanomedicine for the prevention of SARS-CoV-2.

Blood unconjugated bilirubin and tacrolimus are negative predictors of specific cellular immunity in kidney transplant recipients after SAR-CoV-2 inactivated vaccination.

The immunogenicity of SARS-CoV-2 vaccines is poor in kidney transplant recipients (KTRs). The factors related to poor immunogenicity to vaccination in KTRs are not well defined. Here, observational study demonstrated no severe adverse effects were observed in KTRs and healthy participants (HPs) after first or second dose of SARS-CoV-2 inactivated vaccine. Different from HPs with excellent immunity against SARS-CoV-2, IgG antibodies against S1 subunit of spike protein, receptor-binding domain, and nucleocapsid protein were not effectively induced in a majority of KTRs after the second dose of inactivated vaccine. Specific T cell immunity response was detectable in 40% KTRs after the second dose of inactivated vaccine. KTRs who developed specific T cell immunity were more likely to be female, and have lower levels of total bilirubin, unconjugated bilirubin, and blood tacrolimus concentrations. Multivariate logistic regression analysis found that blood unconjugated bilirubin and tacrolimus concentration were significantly negatively associated with SARS-CoV-2 specific T cell immunity response in KTRs. Altogether, these data suggest compared to humoral immunity, SARS-CoV-2 specific T cell immunity response are more likely to be induced in KTRs after administration of inactivated vaccine. Reduction of unconjugated bilirubin and tacrolimus concentration might benefit specific cellular immunity response in KTRs following vaccination.

SARS-CoV-2 induces cardiomyocyte apoptosis and inflammation but can be ameliorated by ACE inhibitor Captopril.

Although the clinical manifestation of COVID-19 is mainly respiratory symptoms, approximately 20% of patients suffer from cardiac complications. COVID-19 patients with cardiovascular disease have higher severity of myocardial injury and poor outcomes. The underlying mechanism of myocardial injury caused by SARS-CoV-2 infection remains unclear. Using a non-transgenic mouse model infected with Beta variant (B.1.351), we found that the viral RNA could be detected in lungs and hearts of infected mice. Pathological analysis showed thinner ventricular wall, disorganized and ruptured myocardial fiber, mild inflammatory infiltration, and mild epicardia or interstitial fibrosis in hearts of infected mice. We also found that SARS-CoV-2 could infect cardiomyocytes and produce infectious progeny viruses in human pluripotent stem cell-derived cardiomyocyte-like cells (hPSC-CMs). SARS-CoV-2 infection caused apoptosis, reduction of mitochondrial integrity and quantity, and cessation of beating in hPSC-CMs. In order to dissect the mechanism of myocardial injury caused by SARS-CoV-2 infection, we employed transcriptome sequencing of hPSC-CMs at different time points after viral infection. Transcriptome analysis showed robust induction of inflammatory cytokines and chemokines, up-regulation of MHC class I molecules, activation of apoptosis signaling and cell cycle arresting. These may cause aggravate inflammation, immune cell infiltration, and cell death. Furthermore, we found that Captopril (hypotensive drugs targeting ACE) treatment could alleviate SARS-CoV-2 induced inflammatory response and apoptosis in cardiomyocytes via inactivating TNF signaling pathways, suggesting Captopril may be beneficial for reducing COVID-19 associated cardiomyopathy. These findings preliminarily explain the molecular mechanism of pathological cardiac injury caused by SARS-CoV-2 infection, providing new perspectives for the discovery of antiviral therapeutics.

Intranasal booster using an Omicron vaccine confers broad mucosal and systemic immunity against SARS-CoV-2 variants.

The highly contagious SARS-CoV-2 Omicron subvariants severely attenuated the effectiveness of currently licensed SARS-CoV-2 vaccines based on ancestral strains administered via intramuscular injection. In this study, we generated a recombinant, replication-incompetent human adenovirus type 5, Ad5-S-Omicron, that expresses Omicron BA.1 spike. Intranasal, but not intramuscular vaccination, elicited spike-specific respiratory mucosal IgA and residential T cell immune responses, in addition to systemic neutralizing antibodies and T cell immune responses against most Omicron subvariants. We tested intranasal Ad5-S-Omicron as a heterologous booster in mice that previously received intramuscular injection of inactivated ancestral vaccine. In addition to inducing serum broadly neutralizing antibodies, there was a significant induction of respiratory mucosal IgA and neutralizing activities against Omicron subvariants BA.1, BA.2, BA.5, BA.2.75, BF.7 as well as pre-Omicron strains Wildtype, Beta, and Delta. Serum and mucosal neutralizing activities against recently emerged XBB, BQ.1, and BQ.1.1 could also be detected but were much lower. Nasal lavage fluids from intranasal vaccination contained multimeric IgA that can bind to at least 10 spike proteins, including Omicron subvariants and pre-Omicron strains, and possessed broadly neutralizing activities. Intranasal vaccination using Ad5-S-Omicron or instillation of intranasal vaccinee's nasal lavage fluids in mouse nostrils protected mice against Omicron challenge. Taken together, intranasal Ad5-S-Omicron booster on the basis of ancestral vaccines can establish effective mucosal and systemic immunity against Omicron subvariants and multiple SARS-CoV-2 variants. This candidate vaccine warrants further development as a safe, effective, and user-friendly infection and transmission-blocking vaccine.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Booster vaccination is required to elicit and maintain COVID-19 vaccine-induced immunity in SIV-infected macaques.

ABSTRACTProlonged infection and possible evolution of SARS-CoV-2 in patients living with uncontrolled HIV-1 infection highlight the importance of an effective vaccination regimen, yet the immunogenicity of COVID-19 vaccines and predictive immune biomarkers have not been well investigated. Herein, we report that the magnitude and persistence of antibody and cell-mediated immunity (CMI) elicited by an Ad5-vectored COVID-19 vaccine are impaired in SIV-infected macaques with high viral loads (> 105 genome copies per ml plasma, SIVhi) but not in macaques with low viral loads (< 105, SIVlow). After a second vaccination, the immune responses are robustly enhanced in all uninfected and SIVlow macaques. These responses also show a moderate increase in 70% SIVhi macaques but decline sharply soon after. Further analysis reveals that decreased antibody and CMI responses are associated with reduced circulating follicular helper T cell (TFH) counts and aberrant CD4/CD8 ratios, respectively, indicating that dysregulation of CD4+ T cells by SIV infection impairs the COVID-19 vaccine-induced immunity. Ad5-vectored COVID-19 vaccine shows no impact on SIV loads or SIV-specific CMI responses. Our study underscores the necessity of frequent booster vaccinations in HIV-infected patients and provides indicative biomarkers for predicting vaccination effectiveness in these patients.

GPI-anchored ligand-BioID2-tagging system identifies Galectin-1 mediating Zika virus entry.

Identification of host factors facilitating pathogen entry is critical for preventing infectious diseases. Here, we report a tagging system consisting of a viral receptor-binding protein (RBP) linked to BioID2, which is expressed on the cell surface via a GPI anchor. Using VSV or Zika virus (ZIKV) RBP, the system (BioID2- RBP(V)-GPI; BioID2-RBP(Z)-GPI) faithfully identifies LDLR and AXL, the receptors of VSV and ZIKV, respectively. Being GPI-anchored is essential for the probe to function properly. Furthermore, BioID2-RBP(Z)-GPI expressed in human neuronal progenitor cells identifies galectin-1 on cell surface pivotal for ZIKV entry. This conclusion is further supported by antibody blocking and galectin-1 silencing in A549 and mouse neural cells. Importantly, Lgals1 -/- mice are significantly more resistant to ZIKV infection than Lgals1 +/+ littermates are, having significantly lower virus titers and fewer pathologies in various organs. This tagging system may have broad applications for identifying protein-protein interactions on the cell surface.

Analysis of B Cell Receptor Repertoires Reveals Key Signatures of the Systemic B Cell Response after SARS-CoV-2 Infection.

A comprehensive study of the B cell response against SARS-CoV-2 could be significant for understanding the immune response and developing therapeutical antibodies and vaccines. To define the dynamics and characteristics of the antibody repertoire following SARS-CoV-2 infection, we analyzed the mRNA transcripts of immunoglobulin heavy chain (IgH) repertoires of 24 peripheral blood samples collected between 3 and 111 days after symptom onset from 10 COVID-19 patients. Massive clonal expansion of naive B cells with limited somatic hypermutation (SHM) was observed in the second week after symptom onset. The proportion of low-SHM IgG clones strongly correlated with spike-specific IgG antibody titers, highlighting the significant activation of naive B cells in response to a novel virus infection. The antibody isotype switching landscape showed a transient IgA surge in the first week after symptom onset, followed by a sustained IgG elevation that lasted for at least 3 months. SARS-CoV-2 infection elicited poly-germ line reactive antibody responses. Interestingly, 17 different IGHV germ line genes recombined with IGHJ6 showed significant clonal expansion. By comparing the IgH repertoires that we sequenced with the 774 reported SARS-CoV-2-reactive monoclonal antibodies (MAbs), 13 shared spike-specific IgH clusters were found. These shared spike-specific IgH clusters are derived from the same lineage of several recently published neutralizing MAbs, including CC12.1, CC12.3, C102, REGN10977, and 4A8. Furthermore, identical spike-specific IgH sequences were found in different COVID-19 patients, suggesting a highly convergent antibody response to SARS-CoV-2. Our analysis based on sequencing antibody repertoires from different individuals revealed key signatures of the systemic B cell response induced by SARS-CoV-2 infection. IMPORTANCE Although the canonical delineation of serum antibody responses following SARS-CoV-2 infection has been well established, the dynamics of antibody repertoire at the mRNA transcriptional level has not been well understood, especially the correlation between serum antibody titers and the antibody mRNA transcripts. In this study, we analyzed the IgH transcripts and characterized the B cell clonal expansion and differentiation, isotype switching, and somatic hypermutation in COVID-19 patients. This study provided insights at the repertoire level for the B cell response after SARS-CoV-2 infection.

Protective humoral and cellular immune responses to SARS-CoV-2 persist up to 1 year after recovery.

SARS-CoV-2 vaccination has been launched worldwide to build effective population-level immunity to curb the spread of this virus. The effectiveness and duration of protective immunity is a critical factor for public health. Here, we report the kinetics of the SARS-CoV-2 specific immune response in 204 individuals up to 1-year after recovery from COVID-19. RBD-IgG and full-length spike-IgG concentrations and serum neutralizing capacity decreases during the first 6-months, but is maintained stably up to 1-year after hospital discharge. Even individuals who had generated high IgG levels during early convalescent stages had IgG levels that had decreased to a similar level one year later. Notably, the RBD-IgG level positively correlates with serum neutralizing capacity, suggesting the representative role of RBD-IgG in predicting serum protection. Moreover, viral-specific cellular immune protection, including spike and nucleoprotein specific, persisted between 6 months and 12 months. Altogether, our study supports the persistence of viral-specific protective immunity over 1 year.

Germline IGHV3-53-encoded RBD-targeting neutralizing antibodies are commonly present in the antibody repertoires of COVID-19 patients.

Monoclonal antibodies (mAbs) encoded by IGHV3-53 (VH3-53) targeting the spike receptor-binding domain (RBD) have been isolated from different COVID-19 patients. However, the existence and prevalence of shared VH3-53-encoded antibodies in the antibody repertoires is not clear. Using antibody repertoire sequencing, we found that the usage of VH3-53 increased after SARS-CoV-2 infection. A highly shared VH3-53-J6 clonotype was identified in 9 out of 13 COVID-19 patients. This clonotype was derived from convergent gene rearrangements with few somatic hypermutations and was evolutionary conserved. We synthesized 34 repertoire-deduced novel VH3-53-J6 heavy chains and paired with a common IGKV1-9 light chain to produce recombinant mAbs. Most of these recombinant mAbs (23/34) possess RBD binding and virus-neutralizing activities, and recognize ACE2 binding site via the same molecular interface. Our computational analysis, validated by laboratory experiments, revealed that VH3-53 antibodies targeting RBD are commonly present in COVID-19 patients' antibody repertoires, indicating many people have germline-like precursor sequences to rapidly generate SARS-CoV-2 neutralizing antibodies. Moreover, antigen-specific mAbs can be digitally obtained through antibody repertoire sequencing and computational analysis.

Mucosal Priming with a Recombinant Influenza A Virus-Vectored Vaccine Elicits T-Cell and Antibody Responses to HIV-1 in Mice.

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Longitudinal Peripheral Blood Transcriptional Analysis Reveals Molecular Signatures of Disease Progression in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients developing severe illness or even death. Disease severity has been associated with increased levels of proinflammatory cytokines and lymphopenia. To elucidate the atlas of peripheral immune response and pathways that might lead to immunopathology during COVID-19 disease course, we performed a peripheral blood RNA sequencing analysis of the same patient's samples collected from symptom onset to full recovery. We found that PBMCs at different disease stages exhibited unique transcriptome characteristics. We observed that SARS-CoV-2 infection caused excessive release of inflammatory cytokines and lipid mediators as well as an aberrant increase of low-density neutrophils. Further analysis revealed an increased expression of RNA sensors and robust IFN-stimulated genes expression but a repressed type I IFN production. SARS-CoV-2 infection activated T and B cell responses during the early onset but resulted in transient adaptive immunosuppression during severe disease state. Activation of apoptotic pathways and functional exhaustion may contribute to the reduction of lymphocytes and dysfunction of adaptive immunity, whereas increase in IL2, IL7, and IL15 may facilitate the recovery of the number and function of lymphocytes. Our study provides comprehensive transcriptional signatures of peripheral blood response in patients with moderate COVID-19.