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 2-3 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 9μg of gp120 in 1010 VLPs). Both envelopes bound to well-characterized 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 to 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.This project received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 681137 to I.B., N.G., A.O., C.B., J.A., R.W.S., and E.Y. It was also partially supported by the Spanish AIDS Research Network (RIS), funded by the Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) “A way to build Europe” (projects RD16CIII/0002/0001, RD16CIII/0002/0005, and RD16CIII/0025/0041), Plan Estatal de I1D1I 2013-2016 to N.G., A.M.M., J.A., V.S.M., E.Y., M.P., A.O., and C.B.; by IDIBAPS to J.M.M. (80:20 Research grant); by the Fondation Dormeur, Vaduz to C.B.; by the Ministerio de Economía, Industria y Competitividad to N.G., V.S.M., and E.Y. (PI17CIII/00049); by the Ministerio de Ciencia e Innovación to N.G., V.S.M., and E.Y. (PI20CIII/00039); by the Consejo Nacional de Innovación, Ciencia y Tecnología to C.B.P.; and by the HHS/ National Institutes of Health (NIH) to C.B. (P01-AI131568).S

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

Permanent control of HIV-1 pathogenesis in exceptional elite controllers: a model of spontaneous cure

Elite controllers (EC) represent a small subset of HIV-1-infected people that spontaneously control viral replication. However, natural virological suppression and absence of immune dysfunction are not always long-term sustained. We define exceptional EC (EEC) as HIV-1 subjects who maintain the EC characteristics without disease progression for more than 25 years. We analyzed three EEC, diagnosed between 1988 and 1992, who never showed signs of clinical disease progression in absence of any antiretroviral treatment. A comprehensive clinical, virological, and immunological study was performed. The individuals simultaneously exhibited ≥3 described host protective alleles, low levels of total HIV-1 DNA (0.50). Inflammation levels of EEC were similar to HIV-1 negative donors. Remarkably, they showed an exceptional lack of viral evolution and 8-fold lower genetic diversity (<0.01 s/n) in env gene than other EC. We postulate that these EEC represent cases of spontaneous functional HIV-1 cure. A non-functional and non-genetically evolving viral reservoir along with an HIV-1-specific immune response seems to be key for the spontaneous functional cure.Work in Centro Nacional de Microbiologia (ISCIII) was supported by grants SAF (2016–77894-R) from Ministerio de Economia y Competitividad (MINECO) (Spain) and Fondo de Investigación Sanitaria (FIS)-Instituto de Salud CarlosIII, grant FIS (PI 13/02269, ISCIII) and in part by the RIS-RETIC grants RD12/0017/0028 and RD16CIII/0002/0005 funded by the ISCIII-FEDER. MP has a contract of RIS-RETIC RD16CIII/0002/0005. This work was supported by grants from the MINECO, FIS-Instituto de Salud CarlosIII, Fondos Europeos para el Desarrollo Regional, FEDER, grant numbers PI16/00684, PI19/01127, CPII014/00025 to ER-M. and FI14/00431 to LT-D.; the Gilead Fellowship Program (grant numbers GLD17/00299); the Red de Investigación en Sida (grant number RD16/0025/0020). ER-M. is supported by Consejería de Salud y Bienestar Social of Junta de Andalucía through the Nicolás Monardes Program (C-0032/17). Research in VS-M group was supported by Fondo de Investigación Sanitaria (FIS)-Instituto de Salud CarlosIII, grant FIS (PI 17CIII/00049). Grifols partially supported work in the AIDS Research Institute IrsiCaixa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

HIV-1 Dual Infected LTNP-EC Patients Developed an Unexpected Antibody Cross-Neutralizing Activity

This study evaluated the neutralization breadth in dually infected (DI) HIV-1 long-term non-progressor elite controller patients (LTNP-EC) using a representative minipanel of 6 viruses from 5 different subtypes. Our results showed an improved neutralization breadth in DI LTNP-EC patients when compared with matched LTNP single-infected patients. The role of viral diversity in neutralization was estimated with the Shannon Entropy and the p-distance in viral quasispecies. We found a positive correlation between neutralization breadth and diversity within the viral quasispecies. This correlation could explain why a group of LTNP-EC patients developed a broad neutralizing response despite having undetectable levels of viremia.Work in CNM is supported by grants from Fundacion para la Investigación del Sida en España (www.fipse.es) and (PI 13/02269) Fondo de Investigación Sanitaria (ISCIII) (www.isciii.es) to CL. Red de Investigación en SIDA www.retic-ris.net to CL. A grant RD12/0017/0036 to CL as part of the Plan Nacional R D+I and cofinanced by ISCIII (www.isciii.es) - Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). CL IDIBAPS work was supported by a grant (RYC-2007-00788) to EY from the Ministerio de Ciencia y Tecnologia www.micinn.es, a grant (PS09/01459) to EY from the Fondo de Investigaciones Sanitarias (ISCIII) (www.isciii.es).S

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

Contains fulltext : 200142.pdf (publisher's version ) (Open Access

Patients characteristics.

<p>nd: not done.</p><p>*: viral load below the limit of detection.</p><p>Patients characteristics.</p

Phylogenetic tree in the C2-V5 <i>env</i> region for LTNP patients and HIV-1 reference viruses.

<p>Sequences in the C2-V5 region in <i>env</i> gene, obtained by limiting dilution, from the three DI patients, the matched single infected individuals LTNP 17–1 (filled dark blue triangle),17–2 (empty dark blue triangle), and 17–3 (light blue triangle) <i>GeneBank accession number KC 595042–595055</i>, LTNP 2–1 (purple triangle), 2–3 (pink triangle) <i>GeneBank accession number KC595056-595080</i>, LTNP 20–1 (green triangle) <i>GeneBank accession numbers (KC595081-2 and KC 595237)</i>, LTNP 21–2 (filled grey triangle), 21–4 (empty grey triangle) and 21–5 (grey diamond) and 21–6 (grey square) (<i>GeneBank accession numbers KC 59546–48</i>, and <i>KT203901</i>-<i>13)</i> and LTNP 3–2 (yellow triangle) (<i>GeneBank accession numbers KT203872-80)</i> and sequences from reference viruses, LTNP and chronic progressor Spanish patients were included in the analysis. Overall, 180 sequences were subjected to phylogenetic analysis using the Maximum Composite Likelihood method in the MEGA5 program. Two sequences from subtype B viruses (89SP061 and HXB2 with <i>GeneBank accession numbers AJ006287 and K03455</i>, respectively) were included (black star). A subtype D sequence (D.CD.83.ELI, <i>GeneBank accession number</i> K03454I) indicated by grey star was also included as out-group. Sequences were obtained from the LANL database (<a href="http://www.hiv.lanl.gov/" target="_blank">http://www.hiv.lanl.gov</a>). Only bootstrap values above 80% are marked. Double infected patients are included in a box and with the following symbols MDM-3 (green circle), MDM-4 (empty green circle), MDM 5 (light green circle),MDM 6 (green square) <i>GeneBank accession numbers KC 594991–595006</i>, LTNP-5–1 (yellow circle), 5–2 (empty yellow square), 5–3 (filled yellow circle), 5–4 (empty yellow diamond), 5–6 (yellow diamond), 5–7 (empty light yellow diamond), 5–9 (yellow square) <i>GeneBank accession numbers K595120-145</i> and <i>KT203881-90</i> LTNP-15-1 (red circle) and 15–3 (empty red circle) (<i>GeneBank accession numbers KT203891-900</i>). Bar corresponds to genetic distance.</p

Clinical parameters and neutralization breadth comparison between Dual vs Single-infected patients.

<p>Six different viruses form 5 different subtypes were used (VI191 subtype A, two subtype B viruses NL4.3 and AC10, 92BR025 subtype C, 92UG024 subtype D, CM244 subtype E). In brackets is represented the subtype and the tier. Numbers indicate the percentage of reduction in infectivity ± SD after neutralization. Neutralization levels are indicated as follows: black square >90%, dark grey square 90–70%, light grey square 70–50% and white square <50% reduction in infectivity. Number of cross-reactive HIV-1-subtypes neutralized by two samples plasma from each patient are represented. Median of cross-reactive HIV-1-neutralizing activity by dual and single infected patient was compared. A statistical analysis was performed with the non-parametric a 2-tailed Mann-Whitney U test with a restrictive significance at the 95% using GraphPad Prism V 4.0 software.</p

Correlation between clinical data and genetic diversity parameters with heterologous NAb response.

<p>Association between viral load (panel A), Cd4+T cells (panel B), years after infection (panel C), Shannon Entropy (panel D), p-distance (panel E), number of synonymous mutations (ds) (panel F), number of non-synonymous mutations (dn) (panel G) and the ratio ds/dn (Panel H) and number of cross-reactive subtypes was calculated. Median of the different clinical data and the genetic parameters for each patient (y axis) was plotted against the number of subtypes neutralized by each the patients (x axis). Patients are identified by the following symbols. LTNP 17 (blue triangle), LTNP 2 (purple triangle), LTNP 20 (green triangle), LTNP 21 (grey triangle), LTNP 3 (orange triangle), MDM (green circle), LTNP-5 (yellow circle), and LTNP 15 (red circle). Correlation between parameters was calculated by linear regression with a 95% of confidence using GraphPad Prism V 4.0 software. For the comparison of the values, a statistical analysis was performed with the non-parametric 2-tailed Mann-Whitney U test with a restrictive significance at the 95% using GraphPad Prism V 4.0 software.</p