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.

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.

A Live-Attenuated Zika Virus Vaccine with High Production Capacity Confers Effective Protection in Neonatal Mice.

Zika virus (ZIKV) infection during pregnancy has been linked to congenital abnormalities, such as microcephaly in infants. An efficacious vaccine is desirable for preventing the potential recurrence of ZIKV epidemic. Here, we report the generation of an attenuated ZIKV (rGZ02a) that has sharply decreased virulence in mice but grows to high titers in Vero cells, a widely approved cell line for manufacturing human vaccines. Compared to the wild-type ZIKV (GZ02) and a plasmid-launched rGZ02p, rGZ02a has 3 unique amino acid alterations in the envelope (E, S304F), nonstructural protein 1 (NS1, R103K), and NS5 (W637R). rGZ02a is more sensitive to type I interferon than GZ02 and rGZ02p, and causes no severe neurological disorders in either wild-type neonatal C57BL/6 mice or type I interferon receptor knockout (Ifnar1-/-) C57BL/6 mice. Immunization with rGZ02a elicits robust inhibitory antibody responses with a certain long-term durability. Neonates born to the immunized dams are effectively protected against ZIKV-caused neurological disorders and brain damage. rGZ02a as a booster vaccine greatly improves the protective immunity primed by Ad2-prME, an adenovirus-vectored vaccine expressing ZIKV prM and E proteins. Our results illustrate that rGZ02a-induced maternal immunity can be transferred to the neonates and confer effective protection. Hence, rGZ02a may be developed as an alternative live-attenuated vaccine and warrants further evaluation. IMPORTANCE Zika virus (ZIKV), a mosquito-borne flavivirus that has caused global outbreaks since 2013, is associated with severe neurological disorders, such as Guillian-Barré syndrome in adults and microcephaly in infants. The ZIKV epidemic has gradually subsided, but a safe and effective vaccine is still desirable to prevent its potential recurrence, especially in countries of endemicity with competent mosquito vectors. Here, we describe a novel live-attenuated ZIKV, rGZ02a, that carries 3 unique amino acid alterations compared to the wild-type GZ02 and a plasmid-launched rGZ02p. The growth capacity of rGZ02a is comparable to GZ02 in Vero cells, but the pathogenicity is significantly attenuated in two mice models. Immunization with rGZ02a elicits robust inhibitory antibody responses in the dams and effectively protects their offspring against ZIKV disease. Importantly, in a heterologous prime-boost regimen, rGZ02a effectively boosts the protective immunity primed by an adenovirus-vectored vaccine. Thus, rGZ02a is a promising candidate for a live-attenuated ZIKV vaccine.

Human Desmoglein-2 and Human CD46 Mediate Human Adenovirus Type 55 Infection, but Human Desmoglein-2 Plays the Major Roles.

Human adenovirus type 55 (HAdV55) represents an emerging respiratory pathogen and causes severe pneumonia with high fatality in humans. The cellular receptors, which are essential for understanding the infection and pathogenesis of HAdV55, remain unclear. In this study, we found that HAdV55 binding and infection were sharply reduced by disrupting the interaction of viral fiber protein with human desmoglein-2 (hDSG2) but only slightly reduced by disrupting the interaction of viral fiber protein with human CD46 (hCD46). Loss-of-function studies using soluble receptors, blocking antibodies, RNA interference, and gene knockout demonstrated that hDSG2 predominantly mediated HAdV55 infection. Nonpermissive rodent cells became susceptible to HAdV55 infection when hDSG2 or hCD46 was expressed, but hDSG2 mediated more efficient HAd55 infection than hCD46. We generated two transgenic mouse lines that constitutively express either hDSG2 or hCD46. Although nontransgenic mice were resistant to HAdV55 infection, infection with HAdV55 was significantly increased in hDSG2+/+ mice but was much less increased in hCD46+/+ mice. Our findings demonstrate that both hDSG2 and hCD46 are able to mediate HAdV55 infection but hDSG2 plays the major roles. The hDSG2 transgenic mouse can be used as a rodent model for evaluation of HAdV55 vaccine and therapeutics.IMPORTANCE Human adenovirus type 55 (HAdV55) has recently emerged as a highly virulent respiratory pathogen and has been linked to severe and even fatal pneumonia in immunocompetent adults. However, the cellular receptors mediating the entry of HAdV55 into host cells remain unclear, which hinders the establishment of HAdV55-infected animal models and the development of antiviral approaches. In this study, we demonstrated that human desmoglein-2 (hDSG2) plays the major roles during HAdV55 infection. Human CD46 (hCD46) could also mediate the infection of HAdV55, but the efficiency was much lower than for hDSG2. We generated two transgenic mouse lines that express either hDSG2 or hCD46, both of which enabled HAd55 infection in otherwise nontransgenic mice. hDSG2 transgenic mice enabled more efficient HAdV55 infection than hCD46 transgenic mice. Our study adds to our understanding of HAdV55 infection and provides an animal model for evaluating HAdV55 vaccines and therapeutics.

Molecular typing and genomic characteristic of human adenoviruses in Datong, Northern China.

The molecular prevalence of human adenoviruses (HAdVs) in Datong city and molecular evolution of HAdV-C species is still obscure. Here, we explored the molecular prevalence of HAdVs by simultaneous sequencing of hexon and fiber. Then, the penton gene fragments of HAdV-C species were determined by sequencing. Finally, genomic and proteotyping analysis were performed for exploration of molecular evolution of unique HAdV-6. Our results showed that dominant molecular types of HAdVs were HAdV-3, HAdV-2, and HAdV-1 based on the hexon and fiber genotype. Among H2F2 isolates, P1H2F2 was most common, followed by P2H2F2 and HAdV-89. The clinical symptoms of HAdV-1 or HAdV-2 infected patients were more severe than HAdV-3 infected patients, the prognosis of HAdV-1, HAdV-2, and HAdV-3 infected patients was indifference. Genomic and proteotyping analysis demonstrated that DT15 was different from HAdV-6 prototype, with high-discrepant sequences localized in the E3 region. In conclusion, HAdV-1 and HAdV-2 have a high affinity to infect younger children and cause more severe symptoms than HAdV-3. The E3 gene of HAdV-C species was considered as highly recombination and mutation region.

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.

Emergence and characterization of a putative novel human adenovirus recombinant HAdV-C104 causing pneumonia in Southern China.

In 2017, a survey of the molecular epidemiology of human adenovirus (HAdV) infections in Southern China based on hexon and fiber genotype demonstrated that the most prevalent genotypes of HAdV were HAdV-3 (n = 62), HAdV-2 (n = 21), and HAdV-7 (n = 16). In addition, two patients were co-infected with two genotypes of HAdV. Interestingly, a novel human adenovirus C recombinant genotype strain was isolated from one of the pneumonia patients in this survey. Phylogenetic, recombination, and proteotyping analysis showed that this novel pathogen originated from the recombination of parental viruses harboring the HAdV-1 penton and hexon gene, and the HAdV-2 fiber gene. It was named 'P1H1F2' and was assigned as HAdV-C104 based on the nomenclature protocol of using three major capsid proteins for characterization. Subsequent in vitro experiments demonstrated that HAdV-C104 had comparable proliferation capacity to HAdV-1, HAdV-2, and another recombination genotype P1H2F2. In addition, the HAdV-C104 infected patient was diagnosed with pneumonia and recovered after antiviral therapy. This report strengthens the hypothesis of recombination as a major pathway for the molecular evolution of HAdV-C species.

Monoclonal Antibodies against Zika Virus NS1 Protein Confer Protection via Fcγ Receptor-Dependent and -Independent Pathways.

Zika virus (ZIKV) infection during pregnancy causes congenital defects such as fetal microcephaly. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) have the potential to suppress ZIKV pathogenicity without enhancement of disease, but the pathways through which they confer protection remain obscure. Here, we report two types of NS1-targeted human MAbs that inhibit ZIKV infection through distinct mechanisms. MAbs 3G2 and 4B8 show a better efficacy than MAb 4F10 in suppressing ZIKV infection in C57BL/6 neonatal mice. Unlike MAb 4F10 that mainly triggers antibody-dependent cell-mediated cytotoxicity (ADCC), MAbs 3G2 and 4B8 not only trigger ADCC but inhibit ZIKV infection without Fcγ receptor-bearing effector cells, possibly at postentry stages. Destroying the Fc-mediated effector function of MAbs 3G2 and 4B8 reduces but does not abolish their protective effects, whereas destroying the effector function of MAb 4F10 eliminates the protective effects, suggesting that MAbs 3G2 and 4B8 engage both Fcγ receptor-dependent and -independent pathways. Further analysis reveals that MAbs 3G2 and 4B8 target the N-terminal region of NS1 protein, whereas MAb 4F10 targets the C-terminal region, implying that the protective efficacy of an NS1-targeted MAb may be associated with its epitope recognition. Our results illustrate that NS1-targeted MAbs have multifaceted protective effects and provide insights for the development of NS1-based vaccines and therapeutics.IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus that has been linked to congenital microcephaly during recent epidemics. No licensed antiviral drug or vaccine is available. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) inhibit ZIKV pathogenicity but do not enhance the disease as envelope protein-targeted MAbs do. However, the protection mechanisms are not fully understood. Here, we show that in the presence or absence of Fcγ receptor-bearing effector cells, NS1-targeted human MAbs 3G2 and 4B8 inhibit ZIKV infection. Compared to MAb 4F10 that has no inhibitory effects without effector cells, 3G2 and 4B8 confer better protection in ZIKV-infected neonatal mice. Destroying the Fc-mediated effector function reduces but does not abolish the protection of 3G2 and 4B8, suggesting that they engage both Fcγ receptor-dependent and -independent pathways. The protective efficacy of NS1-targeted MAbs may be associated with their epitope recognition. Our findings will help to develop NS1-based vaccines and therapeutics.

Corrigendum: Longitudinal Analysis of T and B Cell Receptor Repertoire Transcripts Reveal Dynamic Immune Response in COVID-19 Patients.

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Longitudinal Analysis of T and B Cell Receptor Repertoire Transcripts Reveal Dynamic Immune Response in COVID-19 Patients.

Severe COVID-19 is associated with profound lymphopenia and an elevated neutrophil to lymphocyte ratio. We applied a novel dimer avoidance multiplexed polymerase chain reaction next-generation sequencing assay to analyze T (TCR) and B cell receptor (BCR) repertoires. Surprisingly, TCR repertoires were markedly diminished during the early onset of severe disease but recovered during the convalescent stage. Monitoring TCR repertoires could serve as an indicative biomarker to predict disease progression and recovery. Panoramic concurrent assessment of BCR repertoires demonstrated isotype switching and a transient but dramatic early IgA expansion. Dominant B cell clonal expansion with decreased diversity occurred following recovery from infection. Profound changes in T cell homeostasis raise critical questions about the early events in COVID-19 infection and demonstrate that immune repertoire analysis is a promising method for evaluating emergent host immunity to SARS-CoV-2 viral infection, with great implications for assessing vaccination and other immunological therapies.

Chloroquine inhibits endosomal viral RNA release and autophagy-dependent viral replication and effectively prevents maternal to fetal transmission of Zika virus.

Zika virus (ZIKV) infection can cause neonatal microcephaly and neurological disorders. Currently, there is no designated drug for treating ZIKV infection and preventing neonatal microcephaly. In this study, we evaluated the effect of chloroquine, an anti-malaria drug, in ZIKV infected cells and mouse models. Chloroquine significantly inhibited ZIKV infection in multiple mammalian cell lines. Chloroquine treatment significantly improved the survival of ZIKV-infected 1-day old suckling SCID Beige mice and reduced viremia in adult SCID Beige mice. Importantly, chloroquine protected the fetus from maternal infection by reducing placenta to fetus viral transmission. We found that chloroquine exerts at least two mechanisms in protecting against ZIKV infection: 1) inhibiting endosomal disassembly of the internalized virus and thus reducing the release of viral RNA to the cytoplasm for replication; 2) inhibiting ZIKV RNA replication through blocking ZIKV induced autophagy. Our study suggests that chloroquine treatment warrants to be considered as a mitigation strategy for treating ZIKV infection and preventing ZIKV-associated microcephaly in pregnant women.

Seroprevalence of Neutralizing Antibodies to Human Adenovirus Type 4 and 7 in Healthy Populations From Southern China.

Human adenoviruses type 4 (HAdV4) and 7 (HAdV7) are two major respiratory pathogens and sporadically cause outbreaks of acute respiratory diseases. The neutralizing antibody (nAb) response to these two adenoviruses in civilian populations, which is important for dissecting previous circulations and predicting potential outbreaks, remains largely unknown. In this study, we generated replication-competent HAdV4 and HAdV7 reporter viruses expressing secreted-alkaline-phosphatase (SEAP), and established neutralization assays to investigate the seroprevalence of pre-existing nAb in healthy volunteers from Hunan Province, southern China. The seropositivity rates are 58.4 and 63.8% for anti-HAdV4 nAb and anti-HAdV7 nAb, respectively. High nAb titers (> 1000) were frequently detected in HAdV4-seropositive individuals, whereas most HAdV7-seropositive volunteers had moderate nAb titers (201-1000). The seropositivity rates of anti-HAdV4 nAb and anti-HAdV7 nAb increase with age, with individuals younger than 20 exhibiting the lowest seropositivity rates. Both seropositivity rates and nAb titers are comparable between different sex groups. Notably, HAdV4-seropositive individuals tend to be HAdV7-seropositive and vice versa. Because HAdV4 antisera showed no neutralizing activity to HAdV7 whereas HAdV7 antisera cannot neutralize HAdV4, a subgroup of individuals might be susceptible to infection by HAdV4 and HAdV7 and thus generate nAb to both of them. These results revealed the continuous circulation of HAdV4 and HAdV7 and the lack of protective immunity in more than 35% of people, which emphasized the surveillance of these two HAdVs and the development of prophylactic vaccines.

Hexon and fiber of adenovirus type 14 and 55 are major targets of neutralizing antibody but only fiber-specific antibody contributes to cross-neutralizing activity.

Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development.

Immunization with an adenovirus-vectored TB vaccine containing Ag85A-Mtb32 effectively alleviates allergic asthma.

Current treatments for allergic asthma primarily ameliorate symptoms rather than inhibit disease progression. Regulating the excessive T helper type 2 (Th2) responses may prevent asthma exacerbation. In this study, we investigated the protective effects of Ad5-gsgAM, an adenovirus vector carrying two mycobacterial antigens Ag85A and Mtb32, against allergic asthma. Using an ovalbumin (OVA)-induced asthmatic mouse model, we found that Ad5-gsgAM elicited much more Th1-biased CD4+T and CD8+T cells than bacillus Calmette-Guérin (BCG). After OVA challenge, Ad5-gsgAM-immunized mice showed significantly lowered airway inflammation in comparison with mice immunized with or without BCG. Total serum immunoglobulin E and pulmonary inducible-nitric-oxide-synthase were efficiently reduced. The cytokine profiles in bronchial-alveolar-lavage-fluids (BALFs) were also modulated, as evidenced by the increased level of interferon-γ (IFN-γ) and the decreased level of interleukin (IL)-4, IL-5, and IL-13. Anti-inflammatory cytokine IL-10 was sharply increased, whereas pro-inflammatory cytokine IL-33 was significantly decreased. Importantly, exogenous IL-33 abrogated the protective effects of Ad5-gsgAM, revealing that the suppression of IL-33/ST2 axis substantially contributed to protection against allergic inflammation. Moreover, regulatory T cells were essential for regulating aberrant Th2 responses as well as IL-33/ST2 axis. These results suggested that modulating the IL-33/ST2 axis via adenovirus-vectored mycobacterial antigen vaccination may provide clinical benefits in allergic inflammatory airways disease. KEY MESSAGES: •Ad5-gsgAM elicits Th1 responses and suppresses Th2-mediated allergic asthma in mice. •Ad5-gsgAM inhibits IL-33/ST2 axis by reducing IL-33 secretion but not ILC2 recruiting. •Treg is essential for modulating Th2 responses and IL-33/ST2 axis by Ad5-gsgAM.

Incorporation of NS1 and prM/M are important to confer effective protection of adenovirus-vectored Zika virus vaccine carrying E protein.

Current design of Zika virus (ZIKV) vaccine mainly considered envelope (E) as the major target antigen. Non-structural protein NS1 was seldom considered. Herein, we generated three adenovirus-vectored vaccines carrying E (Ad2-E), or premembrane/membrane (prM/M) with E (Ad2-prME), or NS1 in addition to prM/M with E (Ad2-prME-NS1). Ad2-prME induced higher neutralizing antibody response to ZIKV than Ad2-E, suggesting prM/M is important for the folding of immunogenic E. Most intriguingly, Ad2-prME-NS1 elicited the best viral inhibition when the immune sera were added to ZIKV-infected cells. In ZIKV-challenged neonatal mice born to maternally immunized dams, Ad2-prME-NS1 conferred the best protection in preventing weight loss, neurological disorders, and viral replication. Ad2-prME also conferred significant protection but was less effective than Ad2-prME-NS1, whereas Ad2-E only alleviated neurological symptoms but did not inhibit viral replication. Our study suggested that NS1 should be considered in the design of ZIKV vaccine in addition to prM/M and E.