Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

Study of the cellular and humoral immune responses to SARS-CoV-2 vaccination.

Our understanding of cellular immunity in response to COVID-19 infection or vaccination is limited because of less commonly used techniques. We investigated both the cellular and humoral immune responses before and after the administration of a third dose of the SARS-CoV-2 vaccine among a group of healthcare workers. Cellular immunity was evaluated using the VIDAS interferon-gamma (IFNγ) RUO test, which enables automated measurement of IFNγ levels after stimulating peripheral blood lymphocytes. Booster doses significantly enhanced both cellular and humoral immunity. Concerning cellular response, the booster dose increased the percentage of positive IFNγ release assay (IGRA) results but no difference in IFNγ release was found. The cellular response was not associated with protection against SARS-CoV-2 infection. Interestingly, vaccinated and infected healthcare workers exhibited the highest levels of anti-spike and neutralizing antibodies. In conclusion, the IGRA is a simple method for measuring cellular immune responses after vaccination. However, its usefulness as a complement to the study of humoral responses is yet to be demonstrated in future research.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT- mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy.

BACKGROUND: The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. METHODS: We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. FINDINGS: Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. INTERPRETATION: Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. FUNDING: This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

Evaluation of two anti-SARS-CoV-2 antibody immunoassays for monitoring patients on pre-exposure prophylaxis.

Pre-exposure prophylaxis (PrEP) is crucial to prevent severe COVID-19 in immunocompromised patients. A reliable method is needed to quantify anti-SARS-CoV-2 antibody levels for personalized monitoring during PrEP. We measured the binding antibody concentrations of 63 immunocompromised patients receiving 300mg or 600mg tixagevimab/cilgavimab on PrEP day and twice during the following 3 months. All blood samples were tested using the Abbott anti-SARS-CoV-2 IgG II Quant assay, the Roche Elecsys anti-SARS-CoV-2 S assay, and live virus-based neutralization assays. The results of the two immunoassays were correlated on day 0, 1 month, and 3 months post-PrEP. Passing-Bablok regression demonstrated higher anti-S concentration values measured with the Roche immunoassay compared to those measured with the Abbott immunoassay. Antibody concentrations were higher after 600 mg tixagevimab/cilgavimab prophylaxis than after 300 mg. The neutralizing antibody titers obtained using the omicron BA.5 and BA.2.75 strains were low. Both automated immunoassays are suitable for monitoring immunocompromised patients on PrEP.

Weaker Effects of the Fourth Dose of BNT162b2 SARS-CoV-2 Vaccine on the Elderly Human Population.

The vaccines presently available are less effective in older people due to senescence of their immune systems. We measured the antibody responses of 42 adults living in nursing homes after the third and the fourth doses of an mRNA vaccine and found that the strain (BA.2 and BA.2.75: from 64 to 128, BA.5: from 16 to 32, BQ.1.1: from 16 to 64 among the uninfected) influenced the effect of the fourth dose of vaccine on neutralizing antibodies. The fourth dose also increased binding antibodies (from 1036 BAU/mL to 5371 BAU/mL among the uninfected, from 3700 BAU/mL to 6773 BAU/mL among the BA.5 infected). This effect was less significant than that of the third dose of vaccine for both neutralizing (BA.2: from 8 to 128, BA.5: from 2 to 16, BA.2.75: from 8 to 64, BQ.1.1: from 2 to 16) and binding antibodies (from 139.8 BAU/mL to 2293 BAU/mL). However, the fourth dose attained the 5000 BAU/mL threshold conferring approximately 80% protection against a SARS-CoV-2 BA.2 infection in most individuals, unlike the third.

Whole-genome single molecule real-time sequencing of SARS-CoV-2 Omicron.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.

HIV-1 resistance genotyping by ultra-deep sequencing and 6-month virological response to first-line treatment.

OBJECTIVES: To evaluate the routine use of the Sentosa ultra-deep sequencing (UDS) system for HIV-1 polymerase resistance genotyping in treatment-naïve individuals and to analyse the virological response (VR) to first-line antiretroviral treatment. METHODS: HIV drug resistance was determined on 237 consecutive samples from treatment-naïve individuals using the Sentosa UDS platform with two mutation detection thresholds (3% and 20%). VR was defined as a plasma HIV-1 virus load <50 copies/mL after 6 months of treatment. RESULTS: Resistance to at least one antiretroviral drug with a mutation threshold of 3% was identified in 29% and 16% of samples according to ANRS and Stanford algorithms, respectively. The ANRS algorithm also revealed reduced susceptibility to at least one protease inhibitor (PI) in 14.3% of samples, to one reverse transcriptase inhibitor in 12.7%, and to one integrase inhibitor (INSTI) in 5.1%. For a mutation threshold of 20%, resistance was identified in 24% and 13% of samples according to ANRS and Stanford algorithms, respectively. The 6 months VR was 87% and was similar in the 58% of patients given INSTI-based treatment, in the 16% given PI-based treatment and in the 9% given NNRTI-based treatment. Multivariate analysis indicated that the VR was correlated with the baseline HIV virus load and resistance to at least one PI at both 3% and 20% mutation detection thresholds (ANRS algorithm). CONCLUSIONS: The Vela UDS platform is appropriate for determining antiretroviral resistance in patients on a first-line antiretroviral treatment. Further studies are needed on the use of UDS for therapeutic management.

Influence of Nasopharyngeal Viral Load on the Spread of the Omicron BA.2 Variant.

We used variant typing polymerase chain reaction to describe the evolution of severe acute respiratory syndrome coronavirus 2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing protocols.

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. Single-molecule real-time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole-genome sequencing (WGS) of SARS-CoV-2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3-99.5]) than with the amplicon protocol (99.3% [IQR: 69.9-99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS-CoV-2.

Current immunoassays and detection of antibodies elicited by Omicron SARS-CoV-2 infection.

The present study aimed to determine whether current commercial immunoassays are adequate for detecting anti-Omicron antibodies. We analyzed the anti-SARS-CoV-2 antibody response of 23 unvaccinated individuals 1-2 months after an Omicron infection. All blood samples were tested with a live virus neutralization assay using a clinical Omicron BA.1 strain and four commercial SARS-CoV-2 immunoassays. We assessed three anti-Spike immunoassays (SARS-CoV-2 IgG II Quant [Abbott S], Wantaï anti-SARS-CoV-2 antibody ELISA [Wantaï], Elecsys Anti-SARS-CoV-2 S assay [Roche]) and one anti-Nucleocapsid immunoassay (Abbott SARS-CoV-2 IgG assay [Abbott N]). Omicron neutralizing antibodies were detected in all samples with the live virus neutralization assay. The detection rate of the Abbott S, Wantai, Roche, and Abbott N immunoassays were 65.2%, 69.6%, 86.9%, and 91.3%, respectively. The sensitivities of Abbott S and Wantai immunoassays were significantly lower than that of the live virus neutralization assay (p = 0.004, p = 0.009; Fisher's exact test). Antibody concentrations obtained with anti-S immunoassays were correlated with Omicron neutralizing antibody concentrations. These data provide clinical evidence of the loss of performance of some commercial immunoassays to detect antibodies elicited by Omicron infections. It highlights the need to optimize these assays by adapting antigens to the circulating SARS-CoV-2 strains.

Antibody Titers and Protection against Omicron (BA.1 and BA.2) SARS-CoV-2 Infection.

The emergence of the SARS-CoV-2 variants of concern has greatly influenced the immune correlates of protection, and there are little data about the antibody threshold concentrations to protect against infection with SARS-CoV-2 Omicron BA.1 or BA.2. We analyzed the antibody responses of 259 vaccinated healthcare workers, some of whom had been previously infected by SARS-CoV-2. The median follow-up was 179 days (IQR: 171-182) after blood collection. We detected 88 SARS-CoV-2 Omicron infections during the follow-up period, 55 (62.5%) with SARS-CoV-2 BA.1, and 33 (37.5%) with SARS-CoV-2 BA.2. A neutralizing antibody titer below 8 provided no protection against a BA.1 infection, a titer of 16 or 32 gave 73.2% protection, and a titer of 64 or 128 provided 78.4% protection. Conversely, the BA.2 infection rate did not vary as a function of anti-BA.2 neutralizing antibody titers. Binding antibody concentrations below 6000 BAU/mL provided no protection against Omicron BA.1 infection, 6000-20,000 BAU/mL provided 55.6% protection, and 20,000 or more provided 87.7% protection. There was no difference in BA.2 infection depending on the binding antibody concentration. Further studies are needed to investigate the relationship between antibody concentrations and infection with the Omicron BA.4/5 variants that are becoming predominant worldwide.

Decreased Efficiency of Neutralizing Antibodies from Previously Infected or Vaccinated Individuals against the B.1.617.2 (Delta) SARS-CoV-2 Variant.

The neutralizing antibody response is a key component of adaptive immunity and a primary protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The increased transmissibility of the SARS-CoV-2 Delta variant and its capacity to cause more severe disease could be linked to a significant reduction in neutralizing antibodies generated during a previous infection or vaccination. We analyzed blood samples from 162 unvaccinated health care workers (HCWs) collected 1 to 3 months postinfection and from 263 vaccinated health care workers 1 month after the last injection. We have compared the neutralizing antibody titers obtained using two virus strains, B.1.160 and B.1.617.2 (Delta variant). Binding antibody concentrations were measured by an immunoassay. The median neutralizing antibody titer against the B.1.160 strain was 128 (interquartile range [IQR], 16 to 256) and 32 (IQR, 8 to 128) against the Delta variant. To obtain a neutralizing antibody titer of 32 or 64, a binding antibody concentration of 182 binding antibody units (BAU)/mL (IQR, 81 to 974) was required with the strain B.1.160, while a concentration of 2,595 BAU/mL (IQR, 1,176 to 5,353) was required with the Delta variant. Our data indicate that antibodies neutralize the SARS-CoV-2 Delta variant 4 times less efficiently than they neutralize an earlier strain. Half of the HCWs had decreased protection from 94% to 76.8% or less for the same total antibody concentration. But neutralization might be correlated with other immune responses. The contributions of other responses, such as those of the T cell and B cell systems, to protection require further investigation. IMPORTANCE Recent studies showed that the neutralizing antibody titer is an important contributor to protection against SARS-CoV-2. With the emergence of new variants, the question arises of maintaining the neutralizing capacities of vaccines and/or of a past infection. We had protective data associated with total antibody concentrations and neutralizing antibody titers for a B.1.160 strain. We showed that to maintain the same levels of protection and, therefore, the same levels of neutralizing antibodies, a total antibody concentration 8.5 times greater is required with the Delta strain. (This study has been registered at ClinicalTrials.gov under registration no. NCT04385108.).

Influence of the Delta Variant and Vaccination on the SARS-CoV-2 Viral Load.

Studies comparing SARS-CoV-2 nasopharyngeal (NP) viral load (VL) according to virus variant and host vaccination status have yielded inconsistent results. We conducted a single center prospective study between July and September 2021 at the drive-through testing center of the Toulouse University Hospital. We compared the NP VL of 3775 patients infected by the Delta (n = 3637) and Alpha (n = 138) variants, respectively. Patient's symptoms and vaccination status (2619 unvaccinated, 636 one dose and 520 two doses) were recorded. SARS-CoV-2 RNA testing and variant screening were assessed by using Thermo Fisher® TaqPath™ COVID-19 and ID solutions® ID™ SARS-CoV-2/VOC evolution Pentaplex assays. Delta SARS-CoV-2 infections were associated with higher VL than Alpha (coef = 0.68; p ≤ 0.01) independently of patient's vaccination status, symptoms, age and sex. This difference was higher for patients diagnosed late after symptom onset (coef = 0.88; p = 0.01) than for those diagnosed early (coef = 0.43; p = 0.03). Infections in vaccinated patients were associated with lower VL (coef = -0.18; p ≤ 0.01) independently of virus variant, symptom, age and sex. Our results suggest that Delta infections could lead to higher VL and for a longer period compared to Alpha infections. By effectively reducing the NP VL, vaccination could allow for limiting viral spread, even with the Delta variant.