Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus

Monkeypox has been declared a public health emergency by the World Health Organization.There is an urgent need for efficient and safe vaccines against the monkeypox virus(MPXV)in response to the rapidly spreading monkeypox epidemic.In the age of COVID-19,mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation.Here,we develop two MPXV quadrivalent mRNA vaccines,named mRNA-A-LNP and mRNA-B-LNP,based on two intracellular mature virus specific proteins(A29L and M1R)and two extracellular enveloped virus specific proteins(A35R and B6R).By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice,mice induce MPXV specific IgG antibodies and potent vaccinia virus(VACV)specific neutralizing antibodies.Further,it elicits efficient MPXV specific Th-1 biased cellular immunity,as well as durable effector memory T and germinal center B cell responses in mice.In addition,two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice.And,the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge.Overall,our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics,as well as against outbreaks caused by other orthopoxviruses,including the smallpox virus.

Comparing T-and B-cell responses to COVID-19 vaccines across varied immune backgrounds

The emergence of adapted variants of the SARS-CoV-2 virus has led to a surge in breakthrough infections worldwide.A recent analysis of immune responses in people who received inactivated vaccines has revealed that individuals with no prior infection have limited resistance to Omicron and its sub-lineages,while those with previous infections exhibit a significant amount of neutralizing antibodies and memory B cells.However,specific T-cell responses remain largely unaffected by the mutations,indicating that T-cell-mediated cellular immunity can still provide protection.Moreover,the administration of a third dose of vaccine has resulted in a marked increase in the spectrum and duration of neutralizing antibodies and memory B cells in vivo,which has enhanced resistance to emerging variants such as BA.2.75 and BA.2.12.1.These results highlight the need to consider booster immunization for previously infected individuals and the development of novel vaccination strategies.The rapid spread of adapted variants of the SARS-CoV-2 virus presents a significant challenge to global health.The findings from this study underscore the importance of tailoring vaccination strategies based on individual immune backgrounds and the potential need for booster shots to combat emerging variants.Continued research and development are crucial to discovering new immunization strategies that will effectively protect public health against the evolving virus.

Synthesis and biological evaluation of novel tricyclic matrinic derivatives as potential anti-filovirus agents

Twenty-six novel tricyclic sophoridinic and matrinic derivatives containing a common chlorinated benzene fragment were designed, synthesized and evaluated for their anti-ebolavirus(EBOV)activities. Structure–activity relationship analysis indicated:(i) 12 N-dichlorobenzyl motif was beneficial for the activity;(ii) the chiral configuration at C5 atom might not affect the activity much. Among the target compounds, compound 7d exhibited the most potent potency against EBOV with an IC_(50) value of 5.29 μmol/L and an SI value of over 37.8. Further in vivo anti-EBOV assay of 7d identified its high effectiveness, and in vivo anti-MARV assay of 7d suggested its inspiring broad-spectrum anti-filovirus activity. The results provided powerful information on further strategic optimization and development of this kind of compounds against filoviruses.

Screening and identification of HTNV_(pv)entry inhibitors with high-throughput pseudovirus-based chemiluminescence

Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic pathogens.China has the highest incidence of HFRS,which is mainly caused by HTNV and Seoul virus.No approved antiviral drugs are available for these hantaviral diseases.Here,a chemiluminescence-based highthroughput-screening(HTS)assay was developed and used to screen HTNV pseudovirus(HTNVpv)inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine sources.We identified six compounds with in vitro anti-HTNVpvactivities in the low-micromolar range(EC50values of0.1–2.2μmol/L;selectivity index of 40–900).Among the six selected compounds,cepharanthine not only showed good anti-HTNVpvactivity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 h after infection by94%(180 mg/kg/d,P<0.01),93%(90 mg/kg/d,P<0.01),or 92%(45 mg/kg/d,P<0.01),respectively,in a bioluminescent imaging mouse model.A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry phases.Overall,we have established a HTS method for antiviral drugs screening,and shown that cepharanthine is a candidate for HCPS and HFRS therapy.These findings may offer a starting point for the treatment of patients infected with hantaviruses.

Cross-reactivity of eight SARS-CoV-2 variants rationally predicts immunogenicity clustering in sarbecoviruses

A steep rise in Omicron reinfection cases suggests that this variant has increased immune evasion ability.To evaluate its antigenicity relationship with other variants,antisera from guinea pigs immunized with spike protein of SARS-CoV-2 variants of concern(VOCs)and variants of interest(VOIs)were cross-tested against pseudotyped variants.The neutralization activity against Omicron was markedly reduced when other VOCs or VOIs were used as immunogens,and Omicron(BA.1)-elicited sera did not efficiently neutralize the other variants.However,a Beta or Omicron booster,when administered as the 4th dose 3-months after the 3rd dose of any of the variants,could elicit broad neutralizing antibodies against all of the current variants including Omicron BA.1.Further analysis with 280 available antigen–antibody structures and quantification of immune escape from 715 reported neutralizing antibodies provide explanations for the observed differential immunogenicity.Three distinct clades predicted using an in silico algorithm for clustering of sarbecoviruses based on immune escape provide key information for rational design of vaccines.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment.To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine,the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared.The neutralization activities of Alpha,Beta and Gamma variants’spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant,which were constructed using pseudotyped viruses.Meanwhile,the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants.Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019.Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds,whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold.Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results.This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

Construction of pseudotyped human coronaviruses and detection of pre-existing antibodies in the human population

In order to clarify the pre-exist immunity background of different human coronaviruses(HCoV),this study investigated the positive rate of spike(S)protein antibodies of HCoV,including HCoV-severe acute respiratory syndrome(SARS)-associated coronavirus(SARS-CoV-1),severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),Middle East respiratory syndrome coronavirus(MERS-CoV),HCoV-229E,HCoV-NL63,HCoV-HKU1 and HCoV-OC43,before and after the Coronavirus Disease 2019(COVID-19)outbreak.We utilized pseu-dotyped virus-based neutralization assays(PBNA)or enzyme-linked immunosorbent assays(ELISA)to detect antibody levels against HCoV in serum samples collected in 2009-2010 and 2023.The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative.In the serum samples from 2009 to 2010,neutralizing antibodies against SARS-CoV-2(D614G)were negative,whereas in the serum sam-ples from 2023,73 samples(73%)showed neutralizing reactions with the SARS-CoV-2 D614G strain,96 sam-ples(96%)with the BA.5 strain,and 91 samples(91%)with the BF.7 strain.Among pre-COVID-19 samples,33%(33/100)showed neutralizing reactions with HCoV-229E and 63%(63/100)with HCoV-NL63.Among post-COVID-19 samples,50%(50/100)showed neutralizing reactions with HCoV-229E and 49%(49/100)with HCoV-NL63.Due to the different receptors of alpha coronavirus genus compared to other beta coron-avirus genus,neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by con-structing corresponding pseudotyped virus.Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA.The results revealed that among pre-COVID-19 samples,83%(83/100)and 45%(45/100)had binding activity with HCoV-OC43 and HCoV-HKU1,respectively.Among post-COVID-19 samples,100%(100/100)and 81%(81/100)had binding activity with HCoV-0C43 and HCoV-HKU1,respectively.

Immunological and metabolic characteristics of the Omicron variants infection

The Omicron variants of SARS-CoV-2,primarily authenticated in November 2021 in South Africa,has initiated the 5th wave of global pandemics.Here,we systemically examined immunological and metabolic characteristics of Omicron variants infection.We found Omicron resisted to neutralizing antibody targeting receptor binding domain(RBD)of wildtype SARS-CoV-2.Omicron could hardly be neutralized by sera of Corona Virus Disease 2019(COVID-19)convalescents infected with the Delta variant.Through mass spectrometry on MHC-bound peptidomes,we found that the spike protein of the Omicron variants could generate additional CD8+T cell epitopes,compared with Delta.These epitopes could induce robust CD8+T cell responses.Moreover,we found booster vaccination increased the cross-memory CD8+T cell responses against Omicron.Metabolic regulome analysis of Omicron-specific T cell showed a metabolic profile that promoted the response of memory T cells.Consistently,a greater fraction of memory CD8+T cells existed in Omicron stimulated peripheral blood mononuclear cells(PBMCs).In addition,CD147 was also a receptor for the Omicron variants,and CD147 antibody inhibited infection of Omicron.CD147-mediated Omicron infection in a human CD147 transgenic mouse model induced exudative alveolar pneumonia.Taken together,our data suggested that vaccination booster and receptor blocking antibody are two effective strategies against Omicron.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

To discover new drugs to combat COVID-19,an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed.Here,for the first time,we report the crucial role of cathepsin L(CTSL)in patients with COVID-19.The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity.Correspondingly,SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo,while CTSL overexpression,in turn,enhanced pseudovirus infection in human cells.CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry,as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo.Furthermore,amantadine,a licensed anti-influenza drug,significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo.Therefore,CTSL is a promising target for new anti-COVID-19 drug development.

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has precipitated multiple variants resistant to therapeutic antibodies.In this study,12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries.Of them,two RBD-binding antibodies(F61 and H121)showed high-affinity neutralization against SARS-Co V-2,whereas three S2-target antibodies failed to neutralize SARS-Co V-2.Following structure analysis,F61 identified a linear epitope located in residues G446–S494,which overlapped with angiotensinconverting enzyme 2(ACE2)binding sites,while H121 recognized a conformational epitope located on the side face of RBD,outside from ACE2 binding domain.Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-Co V-2.Importantly,these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351,and reacted with mutations of N501 Y,E484 K,and L452 R,indicated that it may also neutralize the recent India endemic strain B.1.617.The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants,which mitigated the risk of viral escape.Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-Co V-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-Co V-2 variants.

CD147 antibody specifically and effectively inhibits infection and cytokine storm of SARS-CoV-2 and its variants delta,alpha,beta,and gamma

SARS-CoV-2 mutations contribute to increased viral transmissibility and immune escape,compromising the effectiveness of existing vaccines and neutralizing antibodies.An in-depth investigation on COVID-19 pathogenesis is urgently needed to develop a strategy against SARS-CoV-2 variants.Here,we identified CD147 as a universal receptor for SARS-CoV-2 and its variants.Meanwhile,Meplazeumab,a humanized anti-CD147 antibody,could block cellular entry of SARS-CoV-2 and its variants-alpha,beta,gamma,and delta,with inhibition rates of 68.7,75.7,52.1,52.1,and 62.3%at 60μg/ml,respectively.Furthermore,humanized CD147 transgenic mice were susceptible to SARS-CoV-2 and its two variants,alpha and beta.When infected,these mice developed exudative alveolar pneumonia,featured by immune responses involving alveoli-infiltrated macrophages,neutrophils,and lymphocytes and activation of IL-17 signaling pathway.Mechanistically,we proposed that severe COVID-19-related cytokine storm is induced by a"spike protein-CD147-CyPA signaling axis":Infection of SARS-CoV-2 through CD147 initiated the JAK-STAT pathway,which further induced expression of cyclophilin A(CyPA);CyPA reciprocally bound to CD147 and triggered MAPK pathway.Consequently,the MAPK pathway regulated the expression of cytokines and chemokines,which promoted the development of cytokine storm.Importantly,Meplazumab could effectively inhibit viral entry and inflammation caused by SARS-CoV-2 and its variants.Therefore,our findings provided a new perspective for severe COVID-19-related pathogenesis.Furthermore,the validated universal receptor for SARS-CoV-2 and its variants can be targeted for COVID-19 treatment.

Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients

Antibody-dependent cellular cytotoxicity(ADCC)responses to viral infection are a form of antibody regulated immune responses mediated through the Fc fragment.Whether severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)triggered ADCC responses contributes to COVID-19 disease development is currently not well understood.To understand the potential correlation between ADCC responses and COVID-19 disease development,we analyzed the ADCC activity and neutralizing antibody response in 255 individuals ranging from asymptomatic to fatal infections over 1 year post disease.ADCC was elicited by 10 days post-infection,peaked by 11-20 days,and remained detectable until 400 days post-infection.In general,patients with severe disease had higher ADCC activities.Notably,patients who had severe disease and recovered had higher ADCC activities than patients who had severe disease and deceased.Importantly,ADCC activities were mediated by a diversity of epitopes in SARS-COV-2-infected mice and induced to comparable levels against SARS-CoV-2 variants of concern(VOCs)(B.1.1.7,B.1.351,and P.1)as that against the D614G mutant in human patients and vaccinated mice.Our study indicates anti-SARS-CoV-2 ADCC as a major trait of COVID-19 patients with various conditions,which can be applied to estimate the extra-neutralization level against COVID-19,especially lethal COVID-19.

Cellular tropism and antigenicity of mink-derived SARS-CoV-2 variants

Dear Editor,The outbreak of SARS-CoV-2 in minks has been observed recently,raising serious concerns over cross-species transmission and the emergence of variants capable of rendering antibody therapy and vaccines less effective.Here,the species tropism and antigenicity of the spike protein of ten variants were analyzed in pseudovirus-based assays involving 25 cell lines as well as 293T cells expressing ACE2 receptor from 14 species.