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Background: First-generation anti-SARS-CoV-2 monoclonal antibodies (mAbs) used for prophylaxis or therapeutic purposes in immunocompromised patients have been withdrawn because of the emergence of resistant Omicron variants. In 2024, 2 novel mAbs, VYD222/Pemivibart and AZD3152/Sipavibart, were approved by health authorities, but their activity against contemporary JN.1 sublineages is poorly characterized., Methods: We isolated authentic JN.1.1, KP.1.1, LB.1, and KP.3.3 viruses and evaluated their sensitivity to neutralization by these mAbs in 2 target cell lines., Results: Compared to ancestral strains, VYD222/Pemivibart remained moderately active against JN.1 subvariants, with a strong increase of 50% Inhibitory Concentration (IC50), reaching up to 3 to 15 µg/mL for KP3.3. AZD3152/Sipavibart neutralized JN.1.1 but lost antiviral efficacy against KP.1.1, LB.1, and KP3.3., Conclusions: Our results highlight the need for a close clinical monitoring of VYD222/Pemivibart and raise concerns about the clinical efficacy of AZD3152/Sipavibart., Competing Interests: CP, HM, and OS have a patent application for anti-SARS-CoV-2 monoclonal antibodies not used in the present study (PCT/FR2021/070522, WO 2022/228827A1), and HM is a scientific consultant for SpikImm biotech. The remaining authors declare no competing interests., (Copyright © 2024 Pathogens and Immunity.)

Mpox is a zoonotic disease endemic in central and west Africa. However, since 2022, human-adapted mpox virus (MPXV) strains are causing large outbreaks spreading outside these regions, leading the World Health Organization to declare public health emergency twice. Tecovirimat, the most widely used drug to treat these infections, blocks viral egress through a poorly understood mechanism. Tecovirimat-resistant strains, all with mutations in the viral phospholipase F13, pose public health concerns. Herein, we report the structure of an F13 homodimer, both alone and in complex with tecovirimat. We demonstrate that tecovirimat acts as a molecular glue, inducing the dimerization of the phospholipase. F13 escape mutations in MPXV clinical isolates are at the dimer interface and prevent drug-induced dimerization in solution and cells. These findings, which decipher tecovirimat's mode of action, will allow better monitoring of poxvirus outbreaks and pave the way for developing more potent and resilient therapeutics.

DABMA is a chemical molecule optimized from the parent compound ABMA and exhibits broad-spectrum antipathogenic activity by modulating the host's endolysosomal and autophagic pathways. Both DABMA and ABMA inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a cellular assay, which further expands their anti-pathogen spectrum in vitro. However, their precise mechanism of action has not yet been resolved. TMEM175 is a newly characterized endolysosomal channel which plays an essential role in the homeostasis of endosomes and lysosomes as well as organelle fusion. Here, we show that DABMA increases the endosomal TMEM175 current through organelle patch clamping with an EC 50 of 17.9 μm. Depletion of TMEM175 protein significantly decreases the antitoxin activity of DABMA and affects its action on acidic- and Rab7-positive endosomes as well as on endolysosomal trafficking. Thus, TMEM175 is necessary for DABMA's activity and may represent a druggable target for the development of anti-infective drugs. Moreover, DABMA, as an activator of the TMEM175 channel, may be useful for the in-depth characterization of the physiological and pathological roles of this endolysosomal channel., (© 2024 Federation of European Biochemical Societies.)

Antibodies play a pivotal role in protecting from SARS-CoV-2 infection, but their efficacy is challenged by the continuous emergence of viral variants. In this study, we describe two broadly neutralizing antibodies cloned from the memory B cells of a single convalescent individual after infection with ancestral SARS-CoV-2. Cv2.3194, a resilient class 1 anti-RBD antibody, remains active against Omicron sub-variants up to BA.2.86. Cv2.3132, a near pan-Sarbecovirus neutralizer, targets the heptad repeat 2 membrane proximal region. When combined, Cv2.3194 and Cv2.3132 form a complementary SARS-CoV-2 neutralizing antibody cocktail exhibiting a local dose-dependent synergy. Thus, remarkably robust neutralizing memory B cell antibodies elicited in response to ancestral SARS-CoV-2 infection can withstand viral evolution and immune escape. The cooperative effect of such antibody combination may confer a certain level of protection against the latest SARS-CoV-2 variants., Competing Interests: The Institut Pasteur has pending patent applications on “Human neutralizing monoclonal antibodies against SARS-CoV-2 and their use thereof” (PCT/EP2022/058777, WO/2022/228827A1) in which C.P., I.F., T.B., X.M., F.A.R., O.S., and H.M. are inventors, and on “Combined antibodies against Sarbecoviruses and their use thereof” (EP23305528.4, PCT/IB2022/000108) in which C.P., T.B., O.S., and H.M. are inventors, both being licensed by the biotech company SpikImm. H.M. is a scientific consultant for SpikImm, and received consulting fees., (© 2024 The Authors.)

The unceasing circulation of SARS-CoV-2 leads to the continuous emergence of novel viral sublineages. Here, we isolate and characterize XBB.1, XBB.1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1 and JN.1 variants, representing >80% of circulating variants in January 2024. The XBB subvariants carry few but recurrent mutations in the spike, whereas BA.2.86.1 and JN.1 harbor >30 additional changes. These variants replicate in IGROV-1 but no longer in Vero E6 and are not markedly fusogenic. They potently infect nasal epithelial cells, with EG.5.1.3 exhibiting the highest fitness. Antivirals remain active. Neutralizing antibody (NAb) responses from vaccinees and BA.1/BA.2-infected individuals are markedly lower compared to BA.1, without major differences between variants. An XBB breakthrough infection enhances NAb responses against both XBB and BA.2.86 variants. JN.1 displays lower affinity to ACE2 and higher immune evasion properties compared to BA.2.86.1. Thus, while distinct, the evolutionary trajectory of these variants combines increased fitness and antibody evasion., (© 2024. The Author(s).)

Background: SARS-CoV-2 Omicron lineage contains variants with multiple sequence mutations relative to the ancestral strain particularly in the viral spike gene. These mutations are associated inter alia with loss of neutralization sensitivity to sera generated by immunization with vaccines targeting ancestral strains or prior infection with circulating (non-Omicron) variants. Here we present a comparison of vaccine formulation elicited cross neutralization responses using two different assay readouts from a subpopulation of a Phase II/III clinical trial., Methods: Human sera from a Phase II/III trial (NCT04762680) was collected and evaluated for neutralizing responses to SARS-CoV-2 spike antigen protein vaccines formulated with AS03 adjuvant, following a primary series of two-doses of ancestral strain vaccine in individuals who were previously unvaccinated or as an ancestral or variant strain booster vaccine among individuals previously vaccinated with the mRNA BNT162b2 vaccine., Results: We report that a neutralizing response to Omicron BA.1 is induced by the two-dose primary series in 89% of SARS-CoV-2-seronegative individuals. A booster dose of each vaccine formulation raises neutralizing antibody titers that effectively neutralizes Omicron BA.1 and BA.4/5 variants. Responses are highest after the monovalent Beta variant booster and similar in magnitude to human convalescent plasma titers., Conclusion: The findings of this study suggest the possibility to generate greater breadth of cross-neutralization to more recently emerging viral variants through use of a diverged spike vaccine in the form of a Beta variant booster vaccine., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Lingyi Zheng reports a relationship with Sanofi, Swiftwater, PA, USA that includes: employment. Stephen Savarino reports a relationship with Sanofi, Swiftwater, PA, USA that includes: employment. Saranya Sridhar has patent pending to Assignee. Roman M Chicz has patent pending to Assignee. Guy de Bruyn has patent pending to Asignee. Stephen Savarino has patent pending to Assignee. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

SARS-CoV-2 variants with undetermined properties have emerged intermittently throughout the COVID-19 pandemic. Some variants possess unique phenotypes and mutations which allow further characterization of viral evolution and Spike functions. Around 1,100 cases of the B.1.640.1 variant were reported in Africa and Europe between 2021 and 2022, before the expansion of Omicron. Here, we analyzed the biological properties of a B.1.640.1 isolate and its Spike. Compared to the ancestral Spike, B.1.640.1 carried 14 amino acid substitutions and deletions. B.1.640.1 escaped binding by some anti-N-terminal domain and anti-receptor-binding domain monoclonal antibodies, and neutralization by sera from convalescent and vaccinated individuals. In cell lines, infection generated large syncytia and a high cytopathic effect. In primary airway cells, B.1.640.1 replicated less than Omicron BA.1 and triggered more syncytia and cell death than other variants. The B.1.640.1 Spike was highly fusogenic when expressed alone. This was mediated by two poorly characterized and infrequent mutations located in the Spike S2 domain, T859N and D936H. Altogether, our results highlight the cytopathy of a hyper-fusogenic SARS-CoV-2 variant, supplanted upon the emergence of Omicron BA.1. (This study has been registered at ClinicalTrials.gov under registration no. NCT04750720.)IMPORTANCEOur results highlight the plasticity of SARS-CoV-2 Spike to generate highly fusogenic and cytopathic strains with the causative mutations being uncharacterized in previous variants. We describe mechanisms regulating the formation of syncytia and the subsequent consequences in a primary culture model, which are poorly understood., Competing Interests: The authors declare no conflict of interest.

Although anti-severe acute respiratory syndrome-coronavirus 2 antibody kinetics have been described in large populations of vaccinated individuals, we still poorly understand how they evolve during a natural infection and how this impacts viral clearance. For that purpose, we analyzed the kinetics of both viral load and neutralizing antibody levels in a prospective cohort of individuals during acute infection with alpha variant. Using a mathematical model, we show that the progressive increase in neutralizing antibodies leads to a shortening of the half-life of both infected cells and infectious viral particles. We estimated that the neutralizing activity reached 90% of its maximal level within 11 days after symptom onset and could reduce the half-life of both infected cells and circulating virus by a 6-fold factor, thus playing a key role to achieve rapid viral clearance. Using this model, we conducted a simulation study to predict in a more general context the protection conferred by pre-existing neutralization titers, due to either vaccination or prior infection. We predicted that a neutralizing activity, as measured by 50% effective dose > 10 3 , could reduce by 46% the risk of having viral load detectable by standard polymerase chain reaction assays and by 98% the risk of having viral load above the threshold of infectiousness. Our model shows that neutralizing activity could be used to define correlates of protection against infection and transmission., (© 2023 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1 ). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins 2-9 . NL63 uses angiotensin-converting enzyme 2 as a receptor 10 , whereas 229E uses human aminopeptidase-N 11 . HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown 12 . Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Importance: SARS-CoV-2 is a new virus responsible for the Covid-19 pandemic. Although SARS-CoV-2 primarily affects the lungs, other organs are infected. Alterations of testosteronemia and spermatozoa motility in infected men have raised questions about testicular infection, along with high level in the testis of ACE2, the main receptor used by SARS-CoV-2 to enter host cells. Using an organotypic culture of human testis, we found that SARS-CoV-2 replicated with slow kinetics in the testis. The virus first targeted testosterone-producing Leydig cells and then germ-cell nursing Sertoli cells. After a peak followed by the upregulation of antiviral effectors, viral replication in the testis decreased and did not induce any major damage to the tissue. Altogether, our data show that SARS-CoV-2 replicates in the human testis to a limited extent and suggest that testicular damages in infected patients are more likely to result from systemic infection and inflammation than from viral replication in the testis., Competing Interests: The authors declare no conflict of interest.

There have been relatively few small molecules developed with direct activity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two existing antimalarial drugs, pyronaridine and quinacrine, display whole cell activity against SARS-CoV-2 in A549 + ACE2 cells (pretreatment, IC 50 = 0.23 and 0.19 μM, respectively) with moderate cytotoxicity (CC 50 = 11.53 and 9.24 μM, respectively). Moreover, pyronaridine displays in vitro activity against SARS-CoV-2 PL pro (IC 50 = 1.8 μM). Given their existing antiviral activity, these compounds are strong candidates for repurposing against COVID-19 and prompt us to study the structure-activity relationship of the 9-aminoacridine scaffold against SARS-CoV-2 using traditional medicinal chemistry to identify promising new analogs. Our studies identified several novel analogs possessing potent in vitro activity in U2-OS ACE2 GFP 1-10 and 1-11 (IC 50 < 1.0 μM) as well as moderate cytotoxicity (CC 50 > 4.0 μM). Compounds such as 7g , 9c , and 7e were more active, demonstrating selectivity indices SI > 10, and 9c displayed the strongest activity (IC 50 ≤ 0.42 μM, CC 50 ≥ 4.41 μM, SI > 10) among them, indicating that it has potential as a new lead molecule in this series against COVID-19., Competing Interests: The authors declare the following competing financial interest(s): SE is CEO of Collaborations Pharmaceuticals, Inc. TJ, TRL and ACP are employees at Collaborations Pharmaceuticals, Inc. Collaborations Pharmaceuticals, Inc. has obtained FDA orphan drug designations for pyronaridine, tilorone and quinacrine for use against Ebola. CPI has also filed a provisional patent for use of these molecules against Marburg and other viruses. The other authors declare that they have no conflict of interest., (© 2023 The Authors. Published by American Chemical Society.)

Mpox virus (MPXV) caused a multi-country outbreak in non-endemic areas in 2022. Following historic success of smallpox vaccination with vaccinia virus (VACV)-based vaccines, the third generation modified vaccinia Ankara (MVA)-based vaccine was used as prophylaxis for MPXV, but its effectiveness remains poorly characterized. Here, we applied two assays to quantify neutralizing antibodies (NAbs) in sera from control, MPXV-infected, or MVA-vaccinated individuals. Various levels of MVA NAbs were detected after infection, historic smallpox, or recent MVA vaccination. MPXV was minimally sensitive to neutralization. However, addition of complement enhanced detection of responsive individuals and NAb levels. Anti-MVA and -MPXV NAbs were observed in 94% and 82% of infected individuals, respectively, and 92% and 56% of MVA vaccinees, respectively. NAb titers were higher in individuals born before 1980, highlighting the impact of historic smallpox vaccination on humoral immunity. Altogether, our results indicate that MPXV neutralization is complement dependent and uncover mechanisms underlying vaccine effectiveness., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4, and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariant BQ.1.1 became predominant in many countries in December 2022. The subvariants carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lose antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remaine weakly active. BQ.1.1 is also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals are low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increases these titers, which remains about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increases more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitates their spread in immunized populations and raises concerns about the efficacy of most available mAbs., (© 2023. The Author(s).)

The emergence of Omicron sublineages impacts the therapeutic efficacy of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs). Here, we evaluate neutralization and antibody-dependent cellular cytotoxicity (ADCC) activities of 6 therapeutic mAbs against Delta, BA.2, BA.4, and BA.5. The Omicron subvariants escape most antibodies but remain sensitive to bebtelovimab and cilgavimab. Consistent with their shared spike sequence, BA.4 and BA.5 display identical neutralization profiles. Sotrovimab is the most efficient at eliciting ADCC. We also analyze 121 sera from 40 immunocompromised individuals up to 6 months after infusion of Ronapreve (imdevimab + casirivimab) or Evusheld (cilgavimab + tixagevimab). Sera from Ronapreve-treated individuals do not neutralize Omicron subvariants. Evusheld-treated individuals neutralize BA.2 and BA.5, but titers are reduced. A longitudinal evaluation of sera from Evusheld-treated patients reveals a slow decay of mAb levels and neutralization, which is faster against BA.5. Our data shed light on antiviral activities of therapeutic mAbs and the duration of effectiveness of Evusheld pre-exposure prophylaxis., Competing Interests: Declaration of interests T.B., C.P., H.M., and O.S. have a pending patent application for an anti-RBD mAb not used in this study (PCT/FR2021/070522)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Since early 2022, Omicron BA.1 has been eclipsed by BA.2, which was in turn outcompeted by BA.5, which displays enhanced antibody escape properties., Methods: Here, we evaluated the duration of the neutralizing antibody (Nab) response, up to 18 months after Pfizer BNT162b2 vaccination, in individuals with or without BA.1/BA.2 breakthrough infection. We measured neutralization of the ancestral D614G lineage, Delta, and Omicron BA.1, BA.2, and BA.5 variants in 300 sera and 35 nasal swabs from 27 individuals., Findings: Upon vaccination, serum Nab titers were decreased by 10-, 15-, and 25-fold for BA.1, BA.2, and BA.5, respectively, compared with D614G. We estimated that, after boosting, the duration of neutralization was markedly shortened from 11.5 months with D614G to 5.5 months with BA.5. After breakthrough, we observed a sharp increase of Nabs against Omicron subvariants, followed by a plateau and a slow decline after 5-6 months. In nasal swabs, infection, but not vaccination, triggered a strong immunoglobulin A (IgA) response and a detectable Omicron-neutralizing activity., Conclusions: BA.5 spread is partly due to abbreviated vaccine efficacy, particularly in individuals who were not infected with previous Omicron variants., Funding: Work in O.S.'s laboratory is funded by the Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2, ANR Coronamito, and IDISCOVR, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant no. ANR-10-LABX-62-IBEID), HERA european funding and the NIH PICREID (grant no U01AI151758)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The protective immunity against omicron following a BNT162b2 Pfizer booster dose among elderly individuals (ie, those aged >65 years) is not well characterised., Methods: In a community-based, prospective, longitudinal cohort study taking place in France in which 75 residents from three nursing homes were enrolled, we selected 38 residents who had received a two-dose regimen of mRNA vaccine and a booster dose of Pfizer BNT162b2 vaccine. We excluded individuals that did not receive three vaccine doses or did not have available sera samples. We measured anti-S IgG antibodies and neutralisation capacity in sera taken 56 (28-68) and 55 (48-64) days (median (range)) after the 2 nd and 3 rd vaccine doses, respectively. Antibodies targeting the SARS-CoV-2 Spike protein were measured with the S-Flow assay as binding antibody units per milliliter (BAU/mL). Neutralising activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of delta and omicron BA.1., Findings: Among the 38 elderly individuals recruited to the cohort study between November 23 rd , 2020 and April 29 th , 2021, with median age of 88 (range 72-101) years, 30 (78.95%) had been previously infected with SARS-CoV-2. After three vaccine doses, serum neutralising activity was lower against omicron BA.1 (median ED50 of 774.5, range 15.0-34660.0) than the delta variant (median ED50 of 4972.0, range 213.7-66340.0), and higher among previously infected (ie, convalescent; median ED50 against omicron: 1088.0, range 32.6-34660.0) compared with infection-naive residents (median ED50 against omicron: 188.4, range 15.0-8918.0). During the French omicron wave in December 2021-January 2022, 75% (6/8) of naive residents were infected, compared to 25% (7/30) of convalescent residents ( P =0.0114). Anti-Spike antibody levels and neutralising activity against omicron BA.1 after a third BNT162b2 booster dose were lower in those with breakthrough BA.1 infection ( n =13) compared with those without ( n =25), with a median of 1429.9 (range 670.9-3818.3) BAU/mL vs 2528.3 (range 695.4-8832.0) BAU/mL ( P =0.029) and a median ED50 of 281.1 (range 15.0-2136.0) vs 1376.0 (range 32.6-34660.0) ( P =0.0013), respectively., Interpretation: This study shows that elderly individuals who received three vaccine doses elicit neutralising antibodies against the omicron BA.1 variant of SARS-CoV-2. Elderly individuals who had also been previously infected showed higher neutralising activity compared with naive individuals. Yet, breakthrough infections with omicron occurred. Individuals with breakthrough infections had significantly lower neutralising titers compared to individuals without breakthrough infection. Thus, a fourth dose of vaccine may be useful in the elderly population to increase the level of neutralising antibodies and compensate for waning immunity., Funding: Institut Pasteur, Fondation pour la Recherche Médicale (FRM), European Health Emergency Preparedness and Response Authority (HERA), Agence nationale de recherches sur le sida et les hépatites virales - Maladies Infectieuses Emergentes (ANRS-MIE), Agence nationale de la recherche (ANR), Assistance Publique des Hôpitaux de Paris (AP-HP) and Fondation de France., Competing Interests: SvdW is a member of the Data Safety Monitoring Board of the DISCOVERY trial and the EU-solidact trial, and an associate editor within the Eurosurveillance journal. MW and SP declare a pending patent (US 63/057.471), SvdW declares issued patents (EP1697507, EP1694829, PCT/EP2020055939, US16/809.717 and WO20211176099), and a provisional patent (US63003855). All the other authors declare no competing interests., (© 2022 The Authors.)

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in Wuhan COVID-19 convalescents combining serological, cellular, and monoclonal antibody explorations revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor-binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment., (© 2022 Planchais et al.)

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro . Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection., Competing Interests: None of the authors have any financial or other interests related to the submitted work., (© 2022 The Authors.)

The severe acute respiratory syndrome coronavirus 2 Omicron BA.1 sublineage has been supplanted in many countries by the BA.2 sublineage. BA.2 differs from BA.1 by about 21 mutations in its spike. In this study, we first compared the sensitivity of BA.1 and BA.2 to neutralization by nine therapeutic monoclonal antibodies (mAbs). In contrast to BA.1, BA.2 was sensitive to cilgavimab, partly inhibited by imdevimab and resistant to adintrevimab and sotrovimab. We then analyzed sera from 29 immunocompromised individuals up to 1 month after administration of Ronapreve (casirivimab and imdevimab) and/or Evusheld (cilgavimab and tixagevimab) antibody cocktails. All treated individuals displayed elevated antibody levels in their sera, which efficiently neutralized the Delta variant. Sera from Ronapreve recipients did not neutralize BA.1 and weakly inhibited BA.2. Neutralization of BA.1 and BA.2 was detected in 19 and 29 out of 29 Evusheld recipients, respectively. As compared to the Delta variant, neutralizing titers were more markedly decreased against BA.1 (344-fold) than BA.2 (nine-fold). We further report four breakthrough Omicron infections among the 29 individuals, indicating that antibody treatment did not fully prevent infection. Collectively, BA.1 and BA.2 exhibit noticeable differences in their sensitivity to therapeutic mAbs. Anti-Omicron neutralizing activity of Ronapreve and, to a lesser extent, that of Evusheld is reduced in patients' sera., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Decrypting the B cell ontogeny of HIV-1 broadly neutralizing antibodies (bNAbs) is paramount for vaccine design. Here, we characterized IgA and IgG bNAbs of three distinct B cell lineages in a viremic controller, two of which comprised only IgG+ or IgA+ blood memory B cells; the third combined both IgG and IgA clonal variants. 7-269 bNAb in the IgA-only lineage displayed the highest neutralizing capacity despite limited somatic mutation, and delayed viral rebound in humanized mice. bNAbs in all three lineages targeted the N332 glycan supersite. The 2.8-Å resolution cryo-EM structure of 7-269-BG505 SOSIP.664 complex showed a similar pose as 2G12, on an epitope mainly composed of sugar residues comprising the N332 and N295 glycans. Binding and cryo-EM structural analyses showed that antibodies from the two other lineages interact mostly with glycans N332 and N386. Hence, multiple B cell lineages of IgG and IgA bNAbs focused on a unique HIV-1 site of vulnerability can codevelop in HIV-1 viremic controllers., Competing Interests: Disclosures: F.A. Rey is a board member of EureKARE and MELETIUS Therapeutics. No other disclosures were reported., (© 2022 Lorin et al.)

Background: SARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in the UK and had been detected in dozens of countries. It has since then been supplanted by Omicron. The AY.4.2 spike displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain when compared to the original Delta variant (B.1.617.2) and remains poorly characterized., Methods: We compared the Delta and the AY.4.2 spikes, by assessing their binding to antibodies and ACE2 and their fusogenicity. We studied the sensitivity of an authentic AY.4.2 viral isolate to neutralizing antibodies., Findings: The AY.4.2 spike exhibited similar binding to all the antibodies and sera tested, and similar fusogenicity and binding to ACE2 than the ancestral Delta spike. The AY.4.2 virus was slightly less sensitive than Delta to neutralization by a panel of monoclonal antibodies; noticeably, the anti-RBD Imdevimab showed incomplete neutralization. Sensitivity of AY.4.2 to sera from vaccinated individuals was reduced by 1.3 to 3-fold, when compared to Delta., Interpretation: Our results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The spread of AY.4.2 was not due to major changes in spike fusogenicity or ACE2 binding, but more likely to a partially reduced neutralization sensitivity., Funding: The work was funded by Institut Pasteur, Fondation pour la Recherche Médicale, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute, Labex IBEID, ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR., Competing Interests: Declaration of interests C.P., H.M., O.S, T.B., F.R. have a pending patent application for an anti-RBD mAb not used in the present study (PCT/FR2021/070522)., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) are promising molecules for therapeutic or prophylactic interventions. Beyond neutralization, bNAbs exert Fc-dependent functions including antibody-dependent cellular cytotoxicity and activation of the complement. Here, we show that a subset of bNAbs targeting the CD4 binding site and the V1/V2 or V3 loops inhibit viral release from infected cells. We combined immunofluorescence, scanning electron microscopy, transmission electron microscopy and immunogold staining to reveal that some bNAbs form large aggregates of virions at the surface of infected cells. This activity correlates with the capacity of bNAbs to bind to Env at the cell surface and to neutralize cell-free viral particles. We further show that antibody bivalency is required for viral retention, and that aggregated virions are neutralized. We have thus identified an additional antiviral activity of bNAbs, which block HIV-1 release by tethering viral particles at the surface of infected cells., (© 2022. The Author(s).)

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa 1-3 . It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development 4 , and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation., (© 2021 The Authors.)

Background: The dynamics of SARS-CoV-2 alpha variant shedding and immune responses at the nasal mucosa remain poorly characterised., Methods: We measured infectious viral release, antibodies and cytokines in 426 PCR+ nasopharyngeal swabs from individuals harboring non-alpha or alpha variants., Findings: With both lineages, viral titers were variable, ranging from 0 to >10 6  infectious units. Rapid antigenic diagnostic tests were positive in 94% of samples with infectious virus. 68 % of individuals carried infectious virus within two days after onset of symptoms. This proportion decreased overtime. Viable virus was detected up to 14 days. Samples containing anti-spike IgG or IgA did not generally harbor infectious virus. Ct values were slightly but not significantly lower with alpha. This variant was characterized by a fast decrease of infectivity overtime and a marked release of 13 cytokines (including IFN-b, IP-10 and IL-10)., Interpretation: The alpha variant displays modified viral decay and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection., Funding: This retrospective study has been funded by Institut Pasteur, ANRS, Vaccine Research Institute, Labex IBEID, ANR/FRM and IDISCOVR, Fondation pour la Recherche Médicale., Competing Interests: Declaration of Competing Interest L.G., I.S., T.B., F.G.-B., and O.S. have a patent US 63/020,063 entitled “S-Flow: a FACS-based assay for serological analysis of SARS-CoV2 infection” pending. The other authors have no conflict of interests to disclose., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India 1-5 . Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries 6 . The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 and B.1.351 variants were first identified in the United Kingdom and South Africa, respectively, and have since spread to many countries. These variants harboring diverse mutations in the gene encoding the spike protein raise important concerns about their immune evasion potential. Here, we isolated infectious B.1.1.7 and B.1.351 strains from acutely infected individuals. We examined sensitivity of the two variants to SARS-CoV-2 antibodies present in sera and nasal swabs from individuals infected with previously circulating strains or who were recently vaccinated, in comparison with a D614G reference virus. We utilized a new rapid neutralization assay, based on reporter cells that become positive for GFP after overnight infection. Sera from 58 convalescent individuals collected up to 9 months after symptoms, similarly neutralized B.1.1.7 and D614G. In contrast, after 9 months, convalescent sera had a mean sixfold reduction in neutralizing titers, and 40% of the samples lacked any activity against B.1.351. Sera from 19 individuals vaccinated twice with Pfizer Cominarty, longitudinally tested up to 6 weeks after vaccination, were similarly potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Neutralizing titers increased after the second vaccine dose, but remained 14-fold lower against B.1.351. In contrast, sera from convalescent or vaccinated individuals similarly bound the three spike proteins in a flow cytometry-based serological assay. Neutralizing antibodies were rarely detected in nasal swabs from vaccinees. Thus, faster-spreading SARS-CoV-2 variants acquired a partial resistance to neutralizing antibodies generated by natural infection or vaccination, which was most frequently detected in individuals with low antibody levels. Our results indicate that B1.351, but not B.1.1.7, may increase the risk of infection in immunized individuals.

Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation., (© 2020 The Authors.)

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The effect of anti-HIV-1 antibodies on complement activation at the surface of infected cells remains partly understood. Here, we show that a subset of anti-Envelope (Env) broadly neutralizing antibodies (bNAbs), targeting the CD4 binding site and the V3 loop, triggers C3 deposition and complement-dependent cytotoxicity (CDC) on Raji cells engineered to express high surface levels of HIV-1 Env. Primary CD4 T cells infected with laboratory-adapted or primary HIV-1 strains and treated with bNAbs are susceptible to C3 deposition but not to rapid CDC. The cellular protein CD59 and viral proteins Vpu and Nef protect infected cells from CDC mediated by bNAbs or by polyclonal IgGs from HIV-positive individuals. However, complement deposition accelerates the disappearance of infected cells within a few days of culture. Altogether, our results uncover the contribution of complement to the antiviral activity of anti-HIV-1 bNAbs., (© 2019 Institut Pasteur.)

Mucosal immune responses to HIV-1 involve the recognition of the viral envelope glycoprotein (gp)160 by tissue-resident B cells and subsequent secretion of antibodies. To characterize the B cells "sensing" HIV-1 in the gut of infected individuals, we probed monoclonal antibodies produced from single intestinal B cells binding to recombinant gp140 trimers. A large fraction of mucosal B cell antibodies were polyreactive and showed only low affinity to HIV-1 envelope glycoproteins, particularly the gp41 moiety. A few high-affinity gp140 antibodies were isolated but lacked neutralizing, potent ADCC, and transcytosis-blocking capacities. Instead, they displayed cross-reactivity with defined self-antigens. Specifically, intestinal HIV-1 gp41 antibodies targeting the heptad repeat 2 region (HR2) cluster II cross-reacted with the p38α mitogen-activated protein kinase 14 (MAPK14). Hence, physiologic polyreactivity of intestinal B cells and molecular mimicry-based self-reactivity of HIV-1 antibodies are two independent phenomena, possibly diverting and/or impairing mucosal humoral immunity to HIV-1., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death., (© 2017 Institut Pasteur.)

Anti-human immunodeficiency virus type 1 (HIV-1) nonneutralizing antibodies (nnAbs) capable of antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. Broadly neutralizing antibodies (bNAbs) also mediate ADCC in cell culture and rely on their Fc region for optimal efficacy in animal models. Here, we selected 9 monoclonal nnAbs and 5 potent bNAbs targeting various epitopes and conformations of the gp120/41 complex and analyzed the potency of the two types of antibodies to bind and eliminate HIV-1-infected cells in culture. Regardless of their neutralizing activity, most of the selected antibodies recognized and killed cells infected with two laboratory-adapted HIV-1 strains. Some nnAbs also bound bystander cells that may have captured viral proteins. However, in contrast to the bNAbs, the nnAbs bound poorly to reactivated infected cells from 8 HIV-positive individuals and did not mediate effective ADCC against these cells. The nnAbs also inefficiently recognize cells infected with 8 different transmitted-founder (T/F) isolates. The addition of a synthetic CD4 mimetic enhanced the binding and killing efficacy of some of the nnAbs in an epitope-dependent manner without reaching the levels achieved by the most potent bNAbs. Overall, our data reveal important qualitative and quantitative differences between nnAbs and bNAbs in their ADCC capacity and strongly suggest that the breadth of recognition of HIV-1 by nnAbs is narrow. IMPORTANCE Most of the anti-HIV antibodies generated by infected individuals do not display potent neutralizing activities. These nonneutralizing antibodies (nnAbs) with antibody-dependent cellular cytotoxicity (ADCC) have been identified as a protective immune correlate in the RV144 vaccine efficacy trial. However, in primate models, the nnAbs do not protect against simian-human immunodeficiency virus (SHIV) acquisition. Thus, the role of nnAbs with ADCC activity in protection from infection remains debatable. In contrast, broadly neutralizing antibodies (bNAbs) neutralize a large array of viral strains and mediate ADCC in cell culture. We analyzed the capacities of 9 nnAbs and 5 bNAbs to eliminate infected cells. We selected 18 HIV-1 strains, including virus reactivated from the reservoir of HIV-positive individuals and transmitted-founder isolates. We report that the nnAbs bind poorly to cells infected with primary HIV-1 strains and do not mediate potent ADCC. Overall, our data show that the breadth of recognition of HIV-1 by nnAbs is narrow., (Copyright © 2017 American Society for Microbiology.)

The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir.

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

Background: Newly synthesized HIV-1 particles assemble at the plasma membrane of infected cells, before being released as free virions or being transferred through direct cell-to-cell contacts to neighboring cells. Localization of HIV-1 Gag precursor at the cell membrane is necessary and sufficient to trigger viral assembly, whereas the GagPol precursor is additionally required to generate a fully matured virion. HIV-1 Nef is an accessory protein that optimizes viral replication through partly defined mechanisms. Whether Nef modulates Gag and/or GagPol localization and assembly at the membrane and facilitates viral cell-to-cell transfer has not been extensively characterized so far., Results: We report that Nef increases the total amount of Gag proteins present in infected cells, and promotes Gag localization at the cell membrane. Moreover, the processing of p55 into p24 is improved in the presence of Nef. We also examined the effect of Nef during HIV-1 cell-to-cell transfer. We show that without Nef, viral transfer through direct contacts between infected cells and target cells is impaired. With a nef-deleted virus, the number of HIV-1 positive target cells after a short 2h co-culture is reduced, and viral material transferred to uninfected cells is less matured. At later time points, this defect is associated with a reduction in the productive infection of new target cells., Conclusions: Our results highlight a previously unappreciated role of Nef during the viral replication cycle. Nef promotes HIV-1 Gag membrane localization and processing, and facilitates viral cell-to-cell transfer.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.

Dengue viruses (DVs) are responsible for the most medically relevant arboviral diseases. However, the molecular interactions mediating DV entry are poorly understood. We determined that TIM and TAM proteins, two receptor families that mediate the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, serve as DV entry factors. Cells poorly susceptible to DV are robustly infected after ectopic expression of TIM or TAM receptors. Conversely, DV infection of susceptible cells is inhibited by anti-TIM or anti-TAM antibodies or knockdown of TIM and TAM expression. TIM receptors facilitate DV entry by directly interacting with virion-associated PtdSer. TAM-mediated infection relies on indirect DV recognition, in which the TAM ligand Gas6 acts as a bridging molecule by binding to PtdSer within the virion. This dual mode of virus recognition by TIM and TAM receptors reveals how DVs usurp the apoptotic cell clearance pathway for infectious entry., (Copyright © 2012 Elsevier Inc. All rights reserved.)

It has been recognized that macroautophagy constitutes an important survival mechanism that allows both the maintenance of cellular homeostasis and the regulation of programmed cell death pathways (e.g., apoptosis). Although several pathogens have been described to induce autophagy, the prosurvival function of this process in infectious models remains poorly characterized. Our recent studies on chikungunya virus (CHIKV), the causative agent of major epidemics in India, Southeast Asia and southern Europe, reveal a novel mechanism by which autophagy limits the cytopathic effects of CHIKV by impinging upon virus-induced cell death pathways.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.

The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24) impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.