Impact of Dolutegravir plus Lamivudine as First-Line Antiretroviral Treatment on HIV-1 Reservoir and Inflammatory Markers in Peripheral Blood.

OBJECTIVE: To compare the effects of first-line antiretroviral treatment (ART) with dolutegravir plus lamivudine (DTG+3TC) versus DTG plus emtricitabine/tenofovir alafenamide (FTC/TAF) on the evolution of the HIV-1 reservoir and immune activation biomarkers in people with HIV (PWH). METHODS: DUALITY was a 48-week, single-center, randomized, open-label clinical trial in ART-naïve PWH. Participants were randomized (1:1) to receive ART with DTG+3TC (2DR group) or DTG+FTC/TAF (3DR group). Total and intact proviral HIV-1 DNA, cell-associated RNA in CD4+ T cells, the frequency of HIV-infected CD4+ T cells able to produce p24, plasma soluble inflammatory markers (IL-6, sCD14, TRAIL, IP-10, FABP2, CRP and D-dimer), and activation and exhaustion markers in CD4+ and CD8+ T cells were longitudinally determined. RESULTS: Forty-four participants (22 per study arm) were enrolled. Baseline mean (SD) log10 plasma viral load (pVL) and CD4+ T cell counts were 4.4 (0.7) copies/mL and 493 (221) cells/mm3, respectively. All participants completing the study (2DR n=20; 3DR n=21) had pVL <50 copies/mL at week 48, except for one in the 2DR group who was resuppressed after treating syphilis. Changes from baseline to week 48 in all reservoir parameters or in levels of soluble inflammatory biomarkers and activated or exhausted CD4+ and CD8+ T cells were similar between 2DR and 3DR groups. CONCLUSION: First-line ART with DTG+3TC resulted in a similar reduction of HIV-1 persistence parameters in peripheral blood, and comparable changes in immune-associated soluble and T-cell markers compared with DTG+FTC/TAF. These findings support recommendation of DTG/3TC among preferred options for first-line ART in PWH.

This study, named DUALITY, looked at how two different initial HIV treatments affect the amount of hidden HIV in the body and immune system activity. The treatments compared were dolutegravir plus lamivudine (DTG+3TC) and dolutegravir plus emtricitabine/tenofovir alafenamide (DTG+FTC/TAF). The study included 44 people with HIV who had never been treated before. Participants were randomly assigned to one of the two treatments and followed for 48 weeks. The amount of HIV in the participants' CD4+ T cells, as well as markers of immune system activity and inflammation were measured during the study. By the end of the study, almost all participants had their viral load reduced to very low levels, regardless of which treatment they received. Also, there were no significant differences between the two treatment groups in terms of reducing hidden HIV or changes in immune system markers. Overall, the study showed that both treatments were equally effective, supporting the use of DTG+3TC as a preferred option for initial HIV treatment.

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses.

Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus-like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9 µg of gp120 in 1010 VLPs). Both envelopes (Envs) bound to well-characterized broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies (PGT145, VRC01, and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric envelope immunogens induced a comparably strong anti-gp120 response despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. IMPORTANCE It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement in the general population. Here, we show that rabbits immunized with new envelopes (VLP formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.

TL1A-DR3 Plasma Levels Are Predictive of HIV-1 Disease Control, and DR3 Costimulation Boosts HIV-1-Specific T Cell Responses.

Relative control of HIV-1 infection has been linked to genetic and immune host factors. In this study, we analyzed 96 plasma proteome arrays from chronic untreated HIV-1-infected individuals using the classificatory random forest approach to discriminate between uncontrolled disease (plasma viral load [pVL] >50,000 RNA copies/ml; CD4 counts 283 cells/mm3, n = 47) and relatively controlled disease (pVL <10,000 RNA copies/ml; CD4 counts 657 cells/mm3, n = 49). Our analysis highlighted the TNF molecule's relevance, in particular, TL1A (TNFSF15) and its cognate DR3 (TNFSRF25), both of which increased in the relative virus control phenotype. DR3 levels (in plasma and PBMCs) were validated in unrelated cohorts (including long-term nonprogressors), thus confirming their independence from CD4 counts and pVL. Further analysis in combined antiretroviral treatment (cART)-treated individuals with a wide range of CD4 counts (137-1835 cells/mm3) indicated that neither TL1A nor DR3 levels reflected recovery of CD4 counts with cART. Interestingly, in cART-treated individuals, plasma TL1A levels correlated with regulatory T cell frequencies, whereas soluble DR3 was strongly associated with the abundance of effector HLA-DR+CD8+ T cells. A positive correlation was also observed between plasma DR3 levels and the HIV-1-specific T cell responses. In vitro, costimulation of PBMC with DR3-specific mAb increased the magnitude of HIV-1-specific responses. Finally, in splenocytes of DNA.HTI-vaccinated mice, costimulation of HTI peptides and a DR3 agonist (4C12) intensified the magnitude of T cell responses by 27%. These data describe the role of the TL1A-DR3 axis in the natural control of HIV-1 infection and point to the use of DR3 agonists in HIV-1 vaccine regimens.

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery.

Zinc deficiency causes immune dysfunction. In T lymphocytes, hypozincemia promotes thymus atrophy, polarization imbalance, and altered cytokine production. Zinc supplementation is commonly used to boost immune function to prevent infectious diseases in at-risk populations. However, the molecular players involved in zinc homeostasis in lymphocytes are poorly understood. In this paper, we wanted to determine the identity of the transporter responsible for zinc entry into lymphocytes. First, in human Jurkat cells, we characterized the effect of zinc on proliferation and activation and found that zinc supplementation enhances activation when T lymphocytes are stimulated using anti-CD3/anti-CD28 Abs. We show that zinc entry depends on specific pathways to correctly tune the NFAT, NF-κB, and AP-1 activation cascades. Second, we used various human and murine models to characterize the zinc transporter family, Zip, during T cell activation and found that Zip6 was strongly upregulated early during activation. Therefore, we generated a Jurkat Zip6 knockout (KO) line to study how the absence of this transporter affects lymphocyte physiology. We found that although Zip6KO cells showed no altered zinc transport or proliferation under basal conditions, under activation, these KO cells showed deficient zinc transport and a drastically impaired activation program. Our work shows that zinc entry into activated lymphocytes depends on Zip6 and that this transporter is essential for the correct function of the cellular activation machinery.

Mechanisms of Abrupt Loss of Virus Control in a Cohort of Previous HIV Controllers.

Elite and viremic HIV controllers are able to control their HIV infection and maintain undetectable or low-level viremia in the absence of antiretroviral treatment. Despite extensive studies, the immune factors responsible for such exclusive control remain poorly defined. We identified a cohort of 14 HIV controllers that suffered an abrupt loss of HIV control (LoC) to investigate possible mechanisms and virological and immunological events related to the sudden loss of control. The in-depth analysis of these subjects involved the study of cell tropism of circulating virus, evidence for HIV superinfection, cellular immune responses to HIV, as well as an examination of viral adaptation to host immunity by Gag sequencing. Our data demonstrate that a poor capacity of T cells to mediate in vitro viral suppression, even in the context of protective HLA alleles, predicts a loss of viral control. In addition, the data suggest that inefficient viral control may be explained by an increase of CD8 T-cell activation and exhaustion before LoC. Furthermore, we detected a switch from C5- to X4-tropic viruses in 4 individuals after loss of control, suggesting that tropism shift might also contribute to disease progression in HIV controllers. The significantly reduced inhibition of in vitro viral replication and increased expression of activation and exhaustion markers preceding the abrupt loss of viral control may help identify untreated HIV controllers that are at risk of losing control and may offer a useful tool for monitoring individuals during treatment interruption phases in therapeutic vaccine trials.IMPORTANCE A few individuals can control HIV infection without the need for antiretroviral treatment and are referred to as HIV controllers. We have studied HIV controllers who suddenly lose this ability and present with high in vivo viral replication and decays in their CD4+ T-cell counts to identify potential immune and virological factors that were responsible for initial virus control. We identify in vitro-determined reductions in the ability of CD8 T cells to suppress viral control and the presence of PD-1-expressing CD8+ T cells with a naive immune phenotype as potential predictors of in vivo loss of virus control. The findings could be important for the clinical management of HIV controller individuals, and it may offer an important tool to anticipate viral rebound in individuals in clinical studies that include combination antiretroviral therapy (cART) treatment interruptions and which, if not treated quickly, could pose a significant risk to the trial participants.

The extended leader peptide of Haemophilus parasuis trimeric autotransporters conditions their protein expression in Escherichia coli.

Trimeric autotransporters are surface-exposed proteins of Gram-negative bacteria belonging to the type V secretion system. They are involved in virulence and are targets for vaccine and diagnostic tool development, so optimal systems for their expression and purification are required. In the present study, the impact of the extended leader peptide of the Haemophilus parasuis virulence-associated trimeric autotransporters (VtaA) in its production as recombinant proteins in Escherichia coli was evaluated. The 13 genes encoding the VtaA1 to VtaA13 passenger domains of the strain Nagasaki were cloned in the pASK-IBA33plus plasmid and expressed in E. coli. Recombinant protein production was higher for truncated forms in which the entire leader peptide was deleted, and the recombinant protein accumulated in the cytoplasm of the cells. The yield of protein production of the different VtaAs was size dependent, and reached maximal amount at 2-4 h post -induction. The optimization of these conditions allowed to scale-up the production to obtain enough recombinant protein to immunize large animals.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.

UNLABELLED: HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24(Gag) (GQMVHQAIGag140-147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE: Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.

UNLABELLED: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. IMPORTANCE: HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques.

BACKGROUND: None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. METHODS: To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1. RESULTS: Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4(+) and CD8(+) T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4(+) and CD8(+) T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ(+) CD8(+) T cells being Granzyme B(+) and able to degranulate (CD107a(+)). CONCLUSIONS: These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.

Revisiting the taxonomical classification of Porcine Circovirus type 2 (PCV2): still a real challenge.

BACKGROUND: PCV2 has emerged as one of the most devastating viral infections of swine farming, causing a relevant economic impact due to direct losses and control strategies expenses. Epidemiological and experimental studies have evidenced that genetic diversity is potentially affecting the virulence of PVC2. The growing number of PCV2 complete genomes and partial sequences available at GenBank questioned the accepted PCV2 classification. METHODS: Nine hundred seventy five PCV2 complete genomes and 1,270 ORF2 sequences available from GenBank were subjected to recombination, PASC and phylogenetic analyses and results were used for comparison with previous classification scheme. RESULTS: The outcome of these analyses favors the recognition of four genotypes on the basis of ORF2 sequences, namely PCV2a, PCV2b, PCV2c and PCV2d-mPCV2b. To deal with the difficulty of founding an unambiguous classification and accounting the impossibility to define a p-distance cut-off, a set of reference sequences that could be used in further phylogenetic studies for PCV2 genotyping was established. Being aware that extensive phylogenetic analyses are time-consuming and often impracticable during routine diagnostic activity, ORF2 nucleotide positions adequately conserved in the reference sequences were identified and reported to allow a quick genotype differentiation. CONCLUSIONS: Globally, the present work provides an updated scenario of PCV2 genotypes distribution and, based on the limits of the previous classification criteria, proposes new rapid and effective schemes for differentiating the four defined PCV2 genotypes.

Impact of ChAdOx1 or DNA Prime Vaccination on Magnitude, Breadth, and Focus of MVA-Boosted Immunogen-Specific T Cell Responses.

The efficacy of anti-viral T-cell vaccines may greatly depend on their ability to generate high-magnitude responses targeting a broad range of different epitopes. Recently, we created the HIV T-cell immunogen HTI, designed to generate T-cell responses to protein fragments more frequently targeted by HIV controllers. In the present study, we aim to maximize the breadth and magnitude of the T-cell responses generated by HTI by combining different vaccine vectors expressing HTI. We evaluated the ability to induce strong and broad T-cell responses to the HTI immunogen through prime vaccination with DNA plasmid (D) or Chimpanzee Adenovirus Ox1 (ChAdOx1; C) vectors, followed by a Modified Virus Ankara (MVA; M) vaccine boost (DDD, DDDM, C, and CM). HTI-specific T-cell responses after vaccination were measured by IFN-γ-ELISpot assays in two inbred mice strains (C57BL/6 and BALB/c). CM was the schedule triggering the highest magnitude of the response in both mice strains. However, this effect was not reflected in an increase in the breadth of the response but rather in an increase in the magnitude of the response to specific immunodominant epitopes. Immunodominance profiles in the two mouse strains were different, with a clear dominance of T-cell responses to a Pol-derived peptide pool after CM vaccination in C57BL/6. Responses to CM vaccination were also maintained at higher magnitudes over time (13 weeks) compared to other vaccination regimens. Thus, while a ChAdOx1 prime combined with MVA booster vaccination generated stronger and more sustained T-cell responses compared to three DNA vaccinations, the ChAdOx1 primed responses were more narrowly targeted. In conclusion, our findings suggest that the choice of vaccine vectors and prime-boost regimens plays a crucial role in determining the strength, duration, breadth, and focus of T-cell responses, providing further guidance for selecting vaccination strategies.

Immune responses associated with mpox viral clearance in men with and without HIV in Spain: a multisite, observational, prospective cohort study.

BACKGROUND: Since the emergence of the global mpox outbreak in May, 2022, more than 90 000 cases have been diagnosed across 110 countries, disproportionately affecting people with HIV. The durability of mpox-specific immunity is unclear and reinfections have been reported. We aimed to compare mpox immune responses up to 6 months after diagnosis in participants with and without HIV and assess their effect on disease severity and viral clearance dynamics. METHODS: This study was embedded within a prospective, observational, multicentre cohort study of viral clearance dynamics among people with mpox in Spain (MoViE). We included women and men aged 18 years or older, who had signs of mpox, and reported having symptom onset within the previous 10 days at the moment of mpox diagnosis from three sex clinics of the Barcelona metropolitan area. Samples from skin ulcers were collected weekly to estimate the time to clear monkeypox virus (MPXV) from skin lesions. Blood samples were taken at diagnosis, 29, 91, and 182 days later for immune analysis. This included quantifying IgG and IgA against three mpox antigens by ELISA, evaluating in-vitro neutralisation, and characterising mpox-specific T-cell responses using interferon γ detecting enzyme-linked immunospot (ELISpot) assay and multiparametric flow cytometry. FINDINGS: Of the 77 originally enrolled participants, we included 33 participants recruited between July 19, and Oct 6, 2022. Participants without HIV (19 [58%] participants) and participants with HIV (14 [42%] participants) had similar clinical severity and time to MPXV clearance in skin lesions. Participants with HIV had a CD4+ T-cell count median of 777 cells per µL (IQR 484-1533), and 11 (78%) of 14 were virally suppressed on antiretroviral therapy. Nine (27%) of 33 participants were age 49 years or older. 15 (45%) of 33 participants were originally from Spain, and all participants were men. Early humoral responses, particularly concentrations and breadth of IgG and IgA, were associated with milder disease and faster viral clearance. Orthopoxvirus-specific T cells count was also positively correlated with MPXV clearance. Antibody titres declined more rapidly in participants with HIV, but T-cell responses against MPXV were sustained up to day 182 after diagnosis, regardless of HIV status. INTERPRETATION: Higher breadth and magnitude of B-cell and T-cell responses are important in facilitating local viral clearance, limiting mpox dissemination, and reducing disease severity in individuals with preserved immune system. Antibodies appear to contribute to early viral control and T-cell responses are sustained over time, which might contribute to milder presentations during reinfection. FUNDING: Fundació Lluita contra les Infeccions, IrsiCaixa, and Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación e Universidades.

Uncommon pathways of immune escape attenuate HIV-1 integrase replication capacity.

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.

Vaccination with an HIV T-Cell Immunogen (HTI) Using DNA Primes Followed by a ChAdOx1-MVA Boost Is Immunogenic in Gut Microbiota-Depleted Mice despite Low IL-22 Serum Levels.

Despite the important role of gut microbiota in the maturation of the immune system, little is known about its impact on the development of T-cell responses to vaccination. Here, we immunized C57BL/6 mice with a prime-boost regimen using DNA plasmid, the Chimpanzee Adenovirus, and the modified Vaccinia Ankara virus expressing a candidate HIV T-cell immunogen and compared the T-cell responses between individuals with an intact or antibiotic-depleted microbiota. Overall, the depletion of the gut microbiota did not result in significant differences in the magnitude or breadth of the immunogen-specific IFNγ T-cell response after vaccination. However, we observed marked changes in the serum levels of four cytokines after vaccinating microbiota-depleted animals, particularly a significant reduction in IL-22 levels. Interestingly, the level of IL-22 in serum correlated with the abundance of Roseburia in the large intestine of mice in the mock and vaccinated groups with intact microbiota. This short-chain fatty acid (SCFA)-producing bacterium was significantly reduced in the vaccinated, microbiota-depleted group. Therefore, our results indicate that, although microbiota depletion reduces serum levels of IL-22, the powerful vaccine regime used could have overcome the impact of microbiota depletion on IFNγ-producing T-cell responses.

Genomic and antigenic characterization of monomeric autotransporters of Haemophilus parasuis: an ongoing process of reductive evolution.

The genome of the highly pathogenic Haemophilus parasuis Nagasaki strain (serovar 5) was sequenced to 99 % completion. A genomic comparison with two other pathogenic serovar 5 H. parasuis strains identified six genes per genome (bmaA1-bmaA6) encoding ß-barrel monomeric autotransporters, bmaA2 and bmaA3 being pseudogenes in at least one strain. The remaining encoded proteins were predicted to belong to the subtilisin (BmaA1 and BmaA4) and cysteine (BmaA5 and BmaA6) protease families. Allelic polymorphism was detected in other H. parasuis strains by comparative genomic hybridization using microarrays. Recombination events were observed, some of them leading to gene disruption in one of the three strains, although synteny around bmaA genes was conserved. These results suggest that bmaA genes are undergoing a process of reductive evolution. To evaluate their use as potential vaccine antigens, the products of the passenger domains of bmaA1, bmaA4, bmaA5 and bmaA6 were produced in Escherichia coli as recombinant proteins. They were detected by immunoblotting using sera of colostrum-deprived piglets recovering from a sublethal infection with H. parasuis (Nagasaki). The existence of specific antibodies after infection with H. parasuis also demonstrated in vivo expression. Using proteomics, only BmaA6 was detected in the in vitro-grown Nagasaki strain. Interestingly, the translocator domain was found in the outer membrane, while the passenger domain was located in supernatants. These results indicate that BmaA proteins could be considered as immunogen candidates to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be studied further.

Evaluation of the Thermal Stability of a Vaccine Prototype Based on Virus-like Particle Formulated HIV-1 Envelope.

The long-term storage stability of vaccines has a major impact on the roll-out and success of global immunization programs. For the Human Immunodeficiency Virus type 1 (HIV-1) virus-like particle (VLP) vaccine prototype evaluated here, nanoparticle tracking analysis (NTA), and enzyme-linked immunoabsorbent assay (ELISA) results demonstrated a remarkable structural stability. VLPs maintained their integrity and the recognition of relevant B-cell epitopes for three months at 4 and -20 °C. Interestingly, most particles remained intact and preserved the recognition of relevant epitopes even after a week of storage at room temperature.

T-Follicular-Like CD8+ T Cell Responses in Chronic HIV Infection Are Associated With Virus Control and Antibody Isotype Switching to IgG.

T cell responses are considered critical for the in vivo control of HIV, but the contribution of different T cell subsets to this control remains unclear. Using a boosted flow cytometric approach that is able to differentiate CD4+ and CD8+ T cell Th1/Tc1, Th2/Tc2, Th17/Tc17, Treg and Tfh/Tfc-like HIV-specific T cell populations, we identified CD8+ Tfc responses that were related to HIV plasma viral loads and associated with rate of antibody isotype class switching to IgG. This favorable balance towards IgG responses positively correlated with increased virus neutralization, higher avidity of neutralizing antibodies and more potent antibody-dependent cell cytotoxicity (ADCC) in PBMCs from HIV controllers compared to non-controllers. Our results identified the CD8+ Tfc-like T-cell response as a component of effective virus control which could possibly be exploited therapeutically.

Disruption of the HLA-E/NKG2X axis is associated with uncontrolled HIV infections.

The contribution of the HLA-E/NKG2X axis in NK-mediated control of HIV infection remains unclear. We have studied the relationship between HLA-E expression and phenotypical as well as functional characteristics of NK cells, in the context of chronic HIV infection and in an in vitro model of acute infection. High viremia in HIV+ individuals was related to increased HLA-E expression, and changes in NK subpopulations, especially a reduction of the CD56bright as well as an increase in adaptive NK subpopulation. Uncontrolled HIV infection was also characterized by a reversion of the NKG2A/NKG2C expression ratio and a loss of positive and negative regulation of NK mediated by HLA-E. This was reflected in a lower cytotoxic, degranulation and cytokine production capacity, especially in CD56bright and adaptive NK. In line with these results, HLA-E expression showed a positive correlation with viral growth inhibition in an in vitro model of acute infection at day 7, which was lost after 14 days of culture. Using HLA-E expressing K562 cells, we determined that only one out of 11 described HIV-derived HLA-E epitopes increased HLA-E surface stability. In spite of that, eight of the 11 epitopes were capable of increasing degranulation and three drove differences in NK-cell mediated cell lysis or cytokine secretion. In conclusion, our results indicate that HLA-E molecules presenting HIV-derived epitopes may sensitize target cells for NK lysis in early HIV infection. However, prolonged exposure to elevated HLA-E expression levels in vivo may lead to NK cell dysfunction and reduced viral control In chronic infection.

Vaccination with an HIV T-cell immunogen induces alterations in the mouse gut microbiota.

The gut microbiota is emerging as a crucial factor modulating vaccine responses; however, few studies have investigated if vaccines, in turn, can alter the microbiota and to what extent such changes may improve vaccine efficacy. To understand the effect of T-cell vaccination on the gut microbiome, we administered an HIV-1 T-cell immunogen (HTI arm) or PBS (control, Mock arm) to C57Bl/6 mice following a heterologous prime-boost scheme. The longitudinal dynamics of the mice gut microbiota was characterized by 16 S ribosomal RNA sequencing in fecal samples collected from cages, as well as from three gut sections (cecum, small and large intestine). Serum and spleen cells were obtained at the last time point of the study to assess immune correlates using IFNγ ELISPOT and cytokine Luminex® assays. Compared with Mock, HTI-vaccinated mice were enriched in Clostridiales genera (Eubacterium xylanophilum group, Roseburia and Ruminococcus) known as primary contributors of anti-inflammatory metabolites, such as short-chain fatty acids. Such shift was observed after the first HTI dose and remained throughout the study follow-up (18 weeks). However, the enriched Clostridiales genera were different between feces and gut sections. The abundance of bacteria enriched in vaccinated animals positively correlated with HTI-specific T-cell responses and a set of pro-inflammatory cytokines, such as IL-6. This longitudinal analysis indicates that, in mice, T-cell vaccination may promote an increase in gut bacteria known to produce anti-inflammatory molecules, which in turn correlate with proinflammatory cytokines, suggesting an adaptation of the gut microbial milieu to T-cell-induced systemic inflammation.

Does Antigen Glycosylation Impact the HIV-Specific T Cell Immunity?

It is largely unknown how post-translational protein modifications, including glycosylation, impacts recognition of self and non-self T cell epitopes presented by HLA molecules. Data in the literature indicate that O- and N-linked glycosylation can survive epitope processing and influence antigen presentation and T cell recognition. In this perspective, we hypothesize that glycosylation of viral proteins and processed epitopes contribute to the T cell response to HIV. Although there is some evidence for T cell responses to glycosylated epitopes (glyco-epitopes) during viral infections in the literature, this aspect has been largely neglected for HIV. To explore the role of glyco-epitope specific T cell responses in HIV infection we conducted in silico and ex vivo immune studies in individuals with chronic HIV infection. We found that in silico viral protein segments with potentially glycosylable epitopes were less frequently targeted by T cells. Ex vivo synthetically added glycosylation moieties generally masked T cell recognition of HIV derived peptides. Nonetheless, in some cases, addition of simple glycosylation moieties produced neo-epitopes that were recognized by T cells from HIV infected individuals. Herein, we discuss the potential importance of these observations and compare limitations of the employed technology with new methodologies that may have the potential to provide a more accurate assessment of glyco-epitope specific T cell immunity. Overall, this perspective is aimed to support future research on T cells recognizing glycosylated epitopes in order to expand our understanding on how glycosylation of viral proteins could alter host T cell immunity against viral infections.