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.

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.

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.

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.

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.

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.

Priming with Recombinant BCG Expressing HTI Enhances the Magnitude and Breadth of the T-Cell Immune Responses Elicited by MVA.HTI in BALB/c Mice.

The use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a live vaccine vehicle is a promising approach for HIV-1-specific T-cell induction. In this study, we used recombinant BCG expressing HIVACAT T-cell immunogen (HTI), BCG.HTI2auxo.int. BALB/c mice immunization with BCG.HTI2auxo.int prime and MVA.HTI boost was safe and induced HIV-1-specific T-cell responses. Two weeks after boost, T-cell responses were assessed by IFN-γ ELISpot. The highest total magnitude of IFN-γ spot-forming cells (SFC)/106 splenocytes was observed in BCG.HTI2auxo.int primed mice compared to mice receiving MVA.HTI alone or mice primed with BCGwt, although the differences between the vaccination regimens only reached trends. In order to evaluate the differences in the breadth of the T-cell immune responses, we examined the number of reactive peptide pools per mouse. Interestingly, both BCG.HTI2auxo.int and BCGwt primed mice recognized an average of four peptide pools per mouse. However, the variation was higher in BCG.HTI2auxo.int primed mice with one mouse recognizing 11 peptide pools and three mice recognizing few or no peptide pools. The recognition profile appeared to be more spread out for BCG.HTI2auxo.int primed mice and mice only receiving MVA.HTI. Here, we describe a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent infectious diseases.

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.

In-silico drug repurposing study predicts the combination of pirfenidone and melatonin as a promising candidate therapy to reduce SARS-CoV-2 infection progression and respiratory distress caused by cytokine storm.

From January 2020, COVID-19 is spreading around the world producing serious respiratory symptoms in infected patients that in some cases can be complicated by the severe acute respiratory syndrome, sepsis and septic shock, multiorgan failure, including acute kidney injury and cardiac injury. Cost and time efficient approaches to reduce the burthen of the disease are needed. To find potential COVID-19 treatments among the whole arsenal of existing drugs, we combined system biology and artificial intelligence-based approaches. The drug combination of pirfenidone and melatonin has been identified as a candidate treatment that may contribute to reduce the virus infection. Starting from different drug targets the effect of the drugs converges on human proteins with a known role in SARS-CoV-2 infection cycle. Simultaneously, GUILDify v2.0 web server has been used as an alternative method to corroborate the effect of pirfenidone and melatonin against the infection of SARS-CoV-2. We have also predicted a potential therapeutic effect of the drug combination over the respiratory associated pathology, thus tackling at the same time two important issues in COVID-19. These evidences, together with the fact that from a medical point of view both drugs are considered safe and can be combined with the current standard of care treatments for COVID-19 makes this combination very attractive for treating patients at stage II, non-severe symptomatic patients with the presence of virus and those patients who are at risk of developing severe pulmonary complications.

SARS-CoV-2 Consensus-Sequence and Matching Overlapping Peptides Design for COVID19 Immune Studies and Vaccine Development.

Synthetic antigens based on consensus sequences that represent circulating viral isolates are sensitive, time saving and cost-effective tools for in vitro immune monitoring and to guide immunogen design. When based on a representative sequence database, such consensus sequences can effectively be used to test immune responses in exposed and infected individuals at the population level. To accelerate immune studies in SARS-CoV-2 infection, we here describe a SARS-CoV-2 2020 consensus sequence (CoV-2-cons) which is based on more than 1700 viral genome entries in NCBI and encompasses all described SARS-CoV-2 open reading frames (ORF), including recently described frame-shifted and length variant ORF. Based on these sequences, we created curated overlapping peptide (OLP) lists containing between 1500 to 3000 peptides of 15 and 18 amino acids in length, overlapping by 10 or 11 residues, as ideal tools for the assessment of SARS-CoV-2-specific T cell immunity. In addition, CoV-2-cons sequence entropy values are presented along with variant sequences to provide increased coverage of the most variable sections of the viral genome. The identification of conserved protein fragments across the coronavirus family and the corresponding OLP facilitate the identification of T cells potentially cross-reactive with related viruses. This new CoV-2-cons sequence, together with the peptides sets, should provide the basis for SARS-CoV-2 antigen synthesis to facilitate comparability between ex-vivo immune analyses and help to accelerate research on SARS-CoV-2 immunity and vaccine development.

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice.

Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.

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.

Recombinant BCG Expressing HTI Prime and Recombinant ChAdOx1 Boost Is Safe and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

Despite the availability of anti-retroviral therapy, HIV-1 infection remains a massive burden on healthcare systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGΔLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens.

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.

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.

Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant.

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions.

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.

Benzyl-2-Acetamido-2-Deoxy-α-d-Galactopyranoside Increases Human Immunodeficiency Virus Replication and Viral Outgrowth Efficacy In Vitro.

Glycosylation of host and viral proteins is an important posttranslational modification needed to ensure correct function of glycoproteins. For this reason, we asked whether inhibition of O-glycosylation during human immunodeficiency virus (HIV) in vitro replication could affect HIV infectivity and replication rates. We used benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN), a compound that has been widely used to inhibit O-glycosylation in several cell lines. Pretreatment and culture of PHA-blast target cells with BAGN increased the percentage of HIV-infected cells (7.6-fold, p = 0.0115), the per-cell amount of HIV p24 protein (1.3-fold, p = 0.2475), and the viral particles in culture supernatants (7.1-fold, p = 0.0029) compared to BAGN-free cultures. Initiating infection with virus previously grown in the presence of BAGN further increased percentage of infected cells (30-fold, p < 0.0001), intracellular p24 (1.5-fold, p = 0.0433), and secreted viral particles (74-fold, p < 0.0001). BAGN-treated target cells showed less CD25 and CCR5 expression, but increased HLA-DR surface expression, which positively correlated with the number of infected cells. Importantly, BAGN improved viral outgrowth kinetics in 66% of the samples tested, including samples from HIV controllers and subjects in whom no virus could be expanded in the absence of BAGN. Sequencing of the isolated virus indicated no skewing of viral quasi-species populations when compared to BAGN-free culture conditions. BAGN also increased virus production in the ACH2 latency model when used together with latency-reversing agents. Taken together, our results identify BAGN treatment as a simple strategy to improve viral outgrowth in vitro and may provide novel insights into host restriction mechanisms and O-glycosylation-related therapeutic targets for HIV control strategies.

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.