Andreas Mörner

Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein

ABSTRACT Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4+ T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization

Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

ABSTRACT We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.

CCR5 or CXCR4 is required for efficient infection of peripheral blood mononuclear cells by promiscuous human immunodeficiency virus type 2 primary isolates.

Primary human immunodeficiency virus type 2 (HIV-2) isolates are characterized by their ability to use a broad range of coreceptors, including CCR5, CXCR4, and several alternative coreceptors. However, the in vivo relevance of this in vitro promiscuity in coreceptor usage remains unclear. We set out to evaluate the relative importance of CCR5 and CXCR4 for infection of activated peripheral blood mononuclear cells (PBMC). PBMC from donors homozygous for wild-type CCR5 (CCR5(+/+) or CCR5Delta32 (CCR5(-/-)) were tested for their susceptibility to infection with 10 primary HIV-2 isolates with known coreceptor usage by parallel 50% tissue culture infectious dose (TCID50) titrations. Although all isolates, except one, were able to establish productive infection in CCR5(-/-) PBMC, the infection of these cells was inefficient for all isolates that were unable to use CXCR4. For CXCR4-using isolates there were only minor differences in TCID50 between CCR5(+/+) and CCR5(-/-) PBMC. When we compared the replication kinetics in PBMC from donors of the two genotypes we observed an average delay in replication onset of 9 days in the CCR5(-/-) PBMC. This study shows that HIV-2 can use alternative coreceptors for infection of PBMC, but that this infection is much less efficient than infection mediated by CCR5 or CXCR4. Thus, CCR5 and CXCR4 appear to be the major coreceptors for HIV-2 infection of PBMC.

Human neutralizing human immunodeficiency virus type 2-specific Fab molecules generated by phage display

A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.

Immunization with Recombinant HLA Classes I and II, HIV-1 gp140, and SIV p27 Elicits Protection against Heterologous SHIV Infection in Rhesus Macaques

ABSTRACT Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID50) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.

Pandemic influenza A(H1N1)pdm09 seroprevalence in Sweden before and after the pandemic and the vaccination campaign in 2009.

The immunity to pandemic influenza A(H1N1)pdm09 in Sweden before and after the outbreaks in 2009 and 2010 was investigated in a seroepidemiological study. Serum samples were collected at four time points: during 2007 (n = 1968), in October 2009 (n = 2218), in May 2010 (n = 2638) and in May 2011 (n = 2513) and were tested for hemagglutination inhibition (HI) antibodies. In 2007, 4.9% of the population had pre-existing HI titres ≥40, with the highest prevalence (20.0%) in 15–24 year-olds, followed by ≥80 year-olds (9.3%). The overall prevalence of HI titres ≥40 had not changed significantly in October 2009. In May 2010 the prevalence had increased to 48.6% with the highest percentages in 5–14 year-olds (76.2%) andlowest in 75–79 year-olds (18.3%). One year later the prevalence of HI titres ≥40 had increased further to 52.2%. Children 5–14 years had the highest incidence of infection and vaccine uptake as well as the highest post-pandemic protective antibody levels. In contrast, the elderly had high vaccine uptake and low attack rate but low levels of protective antibodies, underlining that factors other than HI antibodies are involved in protection against influenza A(H1N1)pdm09. However, for all age-groups the seroprevalence was stable or increasing between 2010 and 2011, indicating that both vaccine- and infection-induced antibodies were long-lived.

Fine characterization of a V3-region neutralizing epitope in human immunodeficiency virus type 2

We have previously identified two distinct antigenic sites in the third variable region (V3) of human immunodeficiency virus type 2 (HIV-2) corresponding to the principal neutralizing determinant (PND) of HIV-1, the conserved Phe-His-Ser-Gln and Trp-Cys-Arg motifs (positions 315-318 and 329-331), which possibly interact to form a discontinuous antigenic site. The aim of this study was to further identify and characterize the immunogenic sites in the V3-loop of HIV-2 that are important in the binding of neutralizing antibodies and to study in detail the importance of different configurations of peptides corresponding to this region. Peptides representing modifications of the V3-region of HIV-2(SBL6669-ISY) were used for immunization of guinea pigs. With one exception, both the Phe-His-Ser-Gln and the Trp-Cys-Arg motifs were required in the peptide sequences to obtain neutralizing hyperimmune guinea pig sera, and the highest titers were obtained after immunization with 20-27 amino acids (aa) long peptides. Neither substitutions nor deletions of residues between the two motifs, nor the addition of peptide sequences representing a T-helper epitope improved the induction of neutralizing antibodies. Computer simulation modeling revealed that the Phe-315, His-316, Trp-329 and Cys-330 are likely to participate in the formation of a discontinuous epitope. Taken together, these data support the hypothesis that the well conserved motifs FHSQ (positions 315-318) and WCR (positions 329-331) of the HIV-2(SBL6669) V3 region are important targets for neutralizing antibodies, and this may have implications for the design of a future HIV-2 vaccine.

Elevated levels of serum perforin in chronic HIV-1 and acute SIV/SHIV infection.

The impaired functional activity of cytotoxic T lymphocytes and natural killer cells during HIV-1 infection has recently been attributed to decreased intracellular levels of perforin and granzyme B. In sera from individuals chronically infected with HIV-1 we report increased levels of extracellular perforin compared with uninfected individuals. Increased perforin was also observed during experimental SIV/SHIV infection. The combination of reduced intracellular perforin levels and an increased serum level indicates that HIV infection induces aberrant perforin secretion.