Elicitation and enhancement of T and B cell responses

The Major histocompatibility complex class I (MHC-I) has been characterized in such great depth that a number of its key properties are well understood and part of its behavior can even be predicted. It is therefore intriguing that the impact of small epitope modifications on immunogenicity and the elicitation of T-cell repertoires remain often unpredictable and full of surprises. Substitution of the secondary anchor residue at peptide position 3 from a serine to a proline (p3P) significantly increased the stabilization capacity and immunogenicity of the melanoma-associated H-2Db (Db)-restricted epitope gp100 (EGS). Despite this strong enhancement the conformation of the modified epitope (EGP) was not altered and vaccination with EGP generated T-cell responses that recognized cells expressing EGS with high functional avidity. Based on these promising results, the p3P modification was applied to the highly immunodominant Lymphocytic Choriomeningitis Virus epitope gp33 and the associated escape variants Y4F and Y4A. As for gp100, p3P was found to increase the MHC stabilization capacity and immunogenicity of the modified epitopes V3P, PA and PF, while not altering their structures. Accordingly, T-cell responses were cross-reactive between native and p3P enhanced epitopes and, when used for vaccination of C57BL/6 mice, PF elicited a focused T-cell response against Db/Y4F. In parallel, surface plasmon resonance (SPR) measurements revealed that p3P did not only enhance MHC stabilization capacity but also directly increased the affinity of the cognate T-cell receptors (TCRs). To fully characterize the molecular details underlying these two enhancing effects, the thermostability, TCR binding and molecular dynamics (MD) of Db/EGP were measured in comparison with Db/EGS. Furthermore, the contribution of Y159, a highly conserved tyrosine that is structurally juxtaposed to p3P, was assessed using a set of soluble Db-Y159 variants. In conclusion, these measurements clearly demonstrated that specific interactions of p3P with the aromatic ring of Y159 are responsible for the significantly increased MHC stabilization capacity. Surprisingly, the enhanced TCR binding was found to be entirely independent of Y159, suggesting a direct contribution of the buried proline residue to TCR binding. These findings underscore the potential to enhance MHC-I-restricted epitopes at secondary anchor residues, while specifically indicating that proline can directly increase TCR affinity, which could not have been anticipated from our current understanding of the factors shaping TCR recognition. Not entirely different from T-cell elicitation, the induction of broadly neutralizing antibodies against the Human Immunodeficiency Virus type 1 (HIV-1) is to date still an elusive goal despite extensive characterization of the respective antibody-epitope interactions. One of the central challenges is that the virus is highly adapted to immune pressure and the most relevant antibody epitopes on the HIV envelope proteins (Env) are the least immunogenic. Therefore, a highly heterologous prime-boost vaccination strategy was designed in which priming of rabbits with HIV-1 env plasmids was followed by a recombinant Simian Immunodeficiency Virus (SIV) Env boost. While the SIV Env trimers were inherently favorable because of their higher stability, the approach was specifically chosen to preferentially boost antibody responses against the few sites that are conserved in HIV and SIV Env. The described approach was generally validated and warrants future investigations as it lead to the elicitation of potent neutralizing antibodies even though it remains to be fully established if the highly heterologous nature of the prime boost strategy was solely responsible. In summary, the studies presented in this thesis provide the structural and functional platform for a novel and intriguing MHC-I peptide enhancement. Additionally, heterologous immunizations of rabbits offer a promising addition to existing vaccination strategies against HIV

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge

BACKGROUND: Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. PRINCIPAL FINDINGS: HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4(+) T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. CONCLUSIONS: Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4(+) T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10(4) copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4(+) T-cell numbers, low viral load and limited viral divergence

Crystal structure of the HIV-2 neutralizing Fab fragment 7C8 with high specificity to the V3 region of gp125.

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 Å resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell

The deep and narrow antigen-binding site of 7C8, well fitted for elongated epitopes, is highly hydrophobic.

<p>The surface of the antigen-binding site of 7C8, displayed from the perspective of a bound antigen, is colored according to its electrostatic surface potential. To the left, the six CDR loops that form the antigen-binding site are colored according to the sequence-panel. To the right, the relative arrangement and the conformation of the CDRs result in the formation of a deep and narrow highly hydrophobic cleft surrounded by the prominent CDRL1, CDRH1 and CDRH3 loops. Each side of the deep antigen-binding cleft is positively (in blue) and negatively (in red) charged, respectively, while the middle section is highly hydrophobic. A selection of light and heavy chain residues that form the antigen-binding site are indicated in white and black, respectively.</p

The epitope FHSQ is localized centrally on the V3-region of gp125.

<p><b>A</b>) The molecular model of gp125 is very similar to its HIV-1 counterpart gp120. The conserved inner face and the more variable outer face of gp125, which includes the V3 region, are highlighted in grey and blue, respectively. The 15-mer gp125 V3-peptide used to raise 7C8 is displayed in red. The localization and solvent exposure of the epitope GPGR on gp120 and the epitope FHSQ on gp125 are highlighted in orange. <b>B</b>) A putative trimeric model of gp125 displayed with the V3 region oriented towards the target cell indicates the accessibility and solvent exposure of the FHSQ epitope.</p

Overall three-dimensional structure of the HIV-2 neutralizing Fab fragment 7C8.

<p>A) Overall view of the crystal structure of the 7C8 Fab fragment. The light and heavy chains are displayed in light and dark grey, respectively. The six CDR loops are in dark blue (CDRL1), light blue (CDRL2), blue (CDRL3), brown (CDRH1), red (CDRH2) and pink (CDRH3). A black dashed line indicates the disordered loop within the first constant region of the C_H1 domain. B) Stick representation of the stretch of residues 93 to 100C corresponding to the unusually long third complementary determining region of the heavy chain (CDRH3) with the electron density carved around the model at 1.2 σ. <b>C</b>) Ribbon representation of the two monomers found in the assymmetric unit indicating the large variation in elbow angle.</p

Data collection and refinement statistics.

a<p>Number in parentheses indicate the outer-resolution shell.</p>b<p><i>R</i>_merge  =  ∑<i>_hkl</i> ∑<i>_i</i> |<i>I_i</i> (<i>hkl</i>) - 〈<i>I</i> (<i>hkl</i>) 〉|/∑<i>_hkl</i> ∑<i>_i I_i</i> (<i>hkl</i>), where <i>I_i</i>(<i>hkl</i>) is the <i>i</i>th observation of reflection <i>hkl</i> and 〈<i>I</i> (<i>hkl</i>) 〉 is the weighted average intensity for all observations <i>i</i> of reflection hkl.</p>c<p><i>R</i>_cryst  =  Σ_hkl  =  ∑_hkl|<i>F</i>_obs − <i>F</i>_calc|/Σ_hkl |<i>F</i>_obs|.</p>d<p><i>R</i>_free is the same as <i>R</i>_cryst except for 5% of the data excluded from the refinement.</p>e<p>Sum of the TLS and Residual B-factor contributions.</p