Replicative fitness and pathogenicity of primate lentiviruses in lymphoid tissue, primary human and chimpanzee cells: relation to possible jumps to humans.

BACKGROUND: Simian immunodeficiency viruses (SIV) have been jumping between non-human primates in West/Central Africa for thousands of years and yet, the HIV-1 epidemic only originated from a primate lentivirus over 100 years ago. METHODS: This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 22 primate lentiviruses in primary human lymphoid tissue and both primary human and chimpanzee peripheral blood mononuclear cells. FINDINGS: Pairwise competitions revealed that SIV from chimpanzees (cpz) had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for worldwide epidemic. The SIV strains belonging to the "HIV-2 lineage" (including SIVsmm, SIVmac, SIVagm) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains. SIVcpz efficiently replicated in human tonsillar tissue but did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in most chimpanzees. In contrast, HIV-1 isolates and SIV of the HIV-2 lineage were pathogenic to the human tonsillar tissue, almost independent of the level of virus replication. INTERPRETATION: Of all primate lentiviruses, SIV from chimpanzees appears most capable of infecting and replicating in humans, establishing HIV-1. SIV from other Old World monkeys, e.g. the progenitor of HIV-2, replicate slowly in humans due in part to restriction factors. Nonetheless, many of these SIV strains were more pathogenic than SIVcpz. Either SIVcpz evolved into a more pathogenic virus while in humans or a rare SIVcpz, possibly extinct in chimpanzees, was pathogenic immediately following the jump into human. FUNDING: Support for this study to E.J.A. was provided by the NIH/NIAID R01 AI49170 and CIHR project grant 385787. Infrastructure support was provided by the NIH CFAR AI36219 and Canadian CFI/Ontario ORF 36287. Efforts of J.A.B. and N.J.H. was provided by NIH AI099473 and for D.H.C., by VA and NIH AI AI080313.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Inhibition of the lectin pathway of complement activation reduces Acute Respiratory Distress Syndrome severity in a mouse model of SARS-CoV-2 infection.

Most COVID-19 patients requiring ICU care develop an acute respiratory distress syndrome (ARDS), characterised by severe hypoxemia, decreased lung compliance, and high vascular permeability. Activation of the complement system is a hallmark of moderate and severe COVID-19, with abundant deposition of complement proteins reported in inflamed tissue and on the endothelium during COVID-19. Using a transgenic mouse model of SARS-CoV-2 infection we assessed the therapeutic utility of an inhibitory antibody (HG4) targeting the lectin pathway key enzyme MASP-2. Treatment of infected mice with HG4 reduced the disease severity score and improved survival compared to mice that received an isotype control antibody. Administration of HG4 significantly reduced the lung injury score including alveolar inflammatory cell infiltration, alveolar oedema and alveolar haemorrhage. The overall ameliorating effect of MASP-2 inhibition on the severity of COVID-19 pathology is reflected by a significant reduction in the pro-inflammatory activation of brain microglia in HG4 treated mice.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Differential T-cell and antibody responses induced by mRNA versus adenoviral vectored COVID-19 vaccines in patients with immunodeficiencies.

Background: Immunodeficient patients (IDPs) are at higher risk of contracting severe coronavirus disease 2019 (COVID-19). Targeted vaccination strategies have been implemented to enhance vaccine-induced protection. In this population, however, clinical effectiveness is variable and the duration of protection unknown. Objective: We sought to better understand the cellular and humoral immune responses to mRNA and adenoviral vectored COVID-19 vaccines in patients with immunodeficiency. Methods: Immune responses to severe acute respiratory syndrome coronavirus 2 spike were assessed after 2 doses of homologous ChAdOx1-nCoV-19 or BNT162b2 vaccines in 112 infection-naive IDPs and 131 healthy health care workers as controls. Predictors of vaccine responsiveness were investigated. Results: Immune responses to vaccination were low, and virus neutralization by antibody was not detected despite high titer binding responses in many IDPs. In those exhibiting response, the frequency of specific T-cell responses in IDPs was similar to controls, while antibody responses were lower. Sustained vaccine specific differences were identified: T-cell responses were greater in ChAdOx1-nCoV-19- compared to BNT162b2-immunized IDPs, and antibody binding and neutralization were greater in all cohorts immunized with BNT162b2. The positive correlation between T-cell and antibody responses was weak and increased with subsequent vaccination. Conclusion: Immunodeficient patients have impaired immune responses to mRNA and viral vector COVID-19 vaccines that appear to be influenced by vaccine formulation. Understanding the relative roles of T-cell- and antibody-mediated protection as well as the potential of heterologous prime and boost immunization protocols is needed to optimize the vaccination approach in these high-risk groups.

Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design.

To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, we produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA of all influenza A (N1-N9) subtypes and influenza B (B/Victoria and B/Yamagata). These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies and post-vaccination sera with various influenza antigens. We also show that the pELLA is more sensitive than the commercially available NA-Fluor™ in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

Antibody correlates of protection from SARS-CoV-2 reinfection prior to vaccination: A nested case-control within the SIREN study.

OBJECTIVES: To investigate serological differences between SARS-CoV-2 reinfection cases and contemporary controls, to identify antibody correlates of protection against reinfection. METHODS: We performed a case-control study, comparing reinfection cases with singly infected individuals pre-vaccination, matched by gender, age, region and timing of first infection. Serum samples were tested for anti-SARS-CoV-2 spike (anti-S), anti-SARS-CoV-2 nucleocapsid (anti-N), live virus microneutralisation (LV-N) and pseudovirus microneutralisation (PV-N). Results were analysed using fixed effect linear regression and fitted into conditional logistic regression models. RESULTS: We identified 23 cases and 92 controls. First infections occurred before November 2020; reinfections occurred before February 2021, pre-vaccination. Anti-S levels, LV-N and PV-N titres were significantly lower among cases; no difference was found for anti-N levels. Increasing anti-S levels were associated with reduced risk of reinfection (OR 0·63, CI 0·47-0·85), but no association for anti-N levels (OR 0·88, CI 0·73-1·05). Titres >40 were correlated with protection against reinfection for LV-N Wuhan (OR 0·02, CI 0·001-0·31) and LV-N Alpha (OR 0·07, CI 0·009-0·62). For PV-N, titres >100 were associated with protection against Wuhan (OR 0·14, CI 0·03-0·64) and Alpha (0·06, CI 0·008-0·40). CONCLUSIONS: Before vaccination, protection against SARS-CoV-2 reinfection was directly correlated with anti-S levels, PV-N and LV-N titres, but not with anti-N levels. Detectable LV-N titres were sufficient for protection, whilst PV-N titres >100 were required for a protective effect. TRIAL REGISTRATION NUMBER: ISRCTN11041050.

Early and Long-Term HIV-1 Immunogenicity Induced in Macaques by the Combined Administration of DNA, NYVAC and Env Protein-Based Vaccine Candidates: The AUP512 Study.

To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection.

Increased Prevalence of Lassa Fever Virus-Positive Rodents and Diversity of Infected Species Found during Human Lassa Fever Epidemics in Nigeria.

The dynamics of Lassa virus (LASV) infections in rodent reservoirs and their endemic human caseloads remain poorly understood. During the endemic period, human infections are believed to be associated with the seasonal migration of Mastomys natalensis, thought to be the primary reservoir that triggers multiple spillovers of LASV to humans. It has become imperative to improve LASV diagnosis in rodents while updating their prevalence in two regions of Lassa fever endemicity in Nigeria. Rodents (total, 942) were trapped in Ondo (531) and Ebonyi (411) states between October 2018 and April 2020 for detection of LASV using various tissues. Overall, the LASV prevalence was 53.6%. The outbreak area sampled in Ondo had three and two times higher capture success and LASV prevalence, respectively, than Ebonyi State. This correlated with the higher number of annual cases of Lassa fever (LF) in Ondo State versus Ebonyi State. All rodent genera (Mastomys, Rattus, Crocidura, Mus, and Tatera) captured in both states showed slightly variable LASV positivity, with Rattus spp. being the most predominantly infected (77.3%) rodents in Ondo State versus Mastomys spp. (41.6%) in Ebonyi State. The tissues with the highest LASV positivity were the kidneys, spleen, and testes. The finding of a relatively high LASV prevalence in all of the rodent genera captured highlights the complex interspecies transmission dynamics of LASV infections in the reservoirs and their potential association with increased environmental contact, as well as the risk of zoonotic spillover in these communities, which have the highest prevalence of Lassa fever in Nigeria. IMPORTANCE Our findings show the highest LASV positivity in small rodents ever recorded and the first direct detection of LASV in Tatera spp. Our findings also indicate the abundance of LASV-infected small rodents in houses, with probable interspecies transmission through vertical and horizontal coitus routes. Consequently, we suggest that the abundance of different reservoir species for LASV may fuel the epizootic outbreaks of LF in affected human communities. The high prevalence of LASV with the diversity of affected rodents has direct implications for our understanding of the transmission risk, mitigation, and ultimately, the prevention of LF in humans. Optimal tissues for LASV detection in rodents are also presented.

Human seasonal coronavirus neutralization and COVID-19 severity.

The virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global coronavirus disease-2019 (COVID-19) pandemic, spread rapidly around the world causing high morbidity and mortality. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs), and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2. We use functional, neutralizing assays to investigate cross-reactive antibodies and their relationship with COVID-19 severity. We analyzed the neutralization of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63. We found that although HCoV neutralization was very common there was little evidence that these antibodies neutralized SARS-CoV-2. Despite no evidence in cross-neutralization, levels of NL63 neutralizing antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

Secondary Complement Deficiency Impairs Anti-Microbial Immunity to Klebsiella pneumoniae and Staphylococcus aureus During Severe Acute COVID-19.

A high incidence of secondary Klebsiella pneumoniae and Staphylococcus aureus infection were observed in patients with severe COVID-19. The cause of this predisposition to infection is unclear. Our data demonstrate consumption of complement in acute COVID-19 patients reflected by low levels of C3, C4, and loss of haemolytic activity. Given that the elimination of Gram-negative bacteria depends in part on complement-mediated lysis, we hypothesised that secondary hypocomplementaemia is rendering the antibody-dependent classical pathway activation inactive and compromises serum bactericidal activity (SBA). 217 patients with severe COVID-19 were studied. 142 patients suffered secondary bacterial infections. Klebsiella species were the most common Gram-negative organism, found in 58 patients, while S. aureus was the dominant Gram-positive organism found in 22 patients. Hypocomplementaemia was observed in patients with acute severe COVID-19 but not in convalescent survivors three months after discharge. Sera from patients with acute COVID-19 were unable to opsonise either K. pneumoniae or S. aureus and had impaired complement-mediated killing of Klebsiella. We conclude that hyperactivation of complement during acute COVID-19 leads to secondary hypocomplementaemia and predisposes to opportunistic infections.

SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress.

Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.

Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Critical Care Workers Have Lower Seroprevalence of SARS-CoV-2 IgG Compared with Non-patient Facing Staff in First Wave of COVID19.

INTRODUCTION: In early 2020, at first surge of the coronavirus disease 2019 (COVID-19) pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams looking after patients with severe COVID-19. There was considerable anxiety of increased risk of COVID-19 for these staff. To determine whether critical care HCW were at increased risk of hospital acquired infection, we explored the relationship between workplace, patient facing role and evidence of immune exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a quaternary hospital providing a regional critical care response. Routine viral surveillance was not available at this time. METHODS: We screened over 500 HCW (25% of the total workforce) for history of clinical symptoms of possible COVID19, assigning a symptom severity score, and quantified SARS-CoV-2 serum antibodies as evidence of immune exposure to the virus. RESULTS: Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, 14% had evidence of immune exposure. Staffs in patient facing critical care roles were least likely to be seropositive (9%) and staff working in non-patient facing roles most likely to be seropositive (22%). Anosmia and fever were the most discriminating symptoms for seropositive status. Older males presented with more severe symptoms. Of the 12 staff screened positive by nasal swab (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. CONCLUSIONS: Patient facing staff working in critical care do not appear to be at increased risk of hospital acquired infection however the risk of nosocomial infection from non-patient facing staff may be more significant than previous recognised. Most symptoms ascribed to possible COVID-19 were found to have no evidence of immune exposure however seroprevalence may underrepresent infection frequency. Older male staff were at the greatest risk of more severe symptoms.

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.

Cross-Neutralisation of Novel Bombali Virus by Ebola Virus Antibodies and Convalescent Plasma Using an Optimised Pseudotype-Based Neutralisation Assay.

Ebolaviruses continue to pose a significant outbreak threat, and while Ebola virus (EBOV)-specific vaccines and antivirals have been licensed, efforts to develop candidates offering broad species cross-protection are continuing. The use of pseudotyped virus in place of live virus is recognised as an alternative, safer, high-throughput platform to evaluate anti-ebolavirus antibodies towards their development, yet it requires optimisation. Here, we have shown that the target cell line impacts neutralisation assay results and cannot be selected purely based on permissiveness. In expanding the platform to incorporate each of the ebolavirus species envelope glycoprotein, allowing a comprehensive assessment of cross-neutralisation, we found that the recently discovered Bombali virus has a point mutation in the receptor-binding domain which prevents entry into a hamster cell line and, importantly, shows that this virus can be cross-neutralised by EBOV antibodies and convalescent plasma.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection.

We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.