Lennart Åkerblom

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

Presentation of a foreign peptide on the surface of tomato bushy stunt virus.

A 13-amino-acid peptide derived from the V3 loop of human immunodeficiency virus (HIV-1) glycoprotein 120 (gp120) was attached as a C-terminal gene fusion to the coat protein of tomato bushy stunt virus (TBSV). The architecture of this plant virus permitted external display of the foreign sequence 180 times on the surface of the chimaeric virus particle. The chimaera replicated to a level similar to wild-type TBSV and the foreign sequence was retained through six sequential passages in plants. The HIV epitope was detected on the surface of the virus capsid by a V3-specific monoclonal antibody and by human sera from HIV-1-positive patients, demonstrating the potential of using plant-derived chimaeric particles for diagnostic purposes. Chimaeric virus also induced a specific immune response to the foreign HIV epitope when injected into NMRI mice.

Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long‐lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti‐RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear‐cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.

Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response

One of the functions of N-linked glycans of viral glycoproteins is protecting otherwise accessible neutralization epitopes of the viral envelope from neutralizing antibodies. The aim of the present study was to explore the possibility to obtain a more broadly neutralizing immune response by immunizing guinea pigs with gp160 depleted of three N-linked glycans in the CD4-binding domain by site-directed mutagenesis. Mutant and wild type gp160 were formulated into immunostimulating complexes and injected s.c. into guinea pigs. Both preparations induced high serum antibody response to native gp120 and V3 peptides. Both preparations also induced antibodies that bound equally well to the V3 loop or the CD4-binding region, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The sera from animals, immunized with mutated glycoprotein, did not neutralize nonrelated HIV strains better than did sera from animals, immunized with wild type glycoprotein. Instead, a pattern of preferred homologous neutralization was observed, i.e., sera from animals, immunized with mutant gp160, neutralized mutant virus better than wild type virus, and vice versa. These data indicated that elimination of the three N-linked glycans from gp160 resulted in an altered local antigenic conformation but did not uncover hidden neutralization epitopes, broadening the immune response.

Neutralizing Antibody Response During Human Immunodeficiency Virus Type 1 Infection: Type and Group Specificity and Viral Escape

The paradox that group-specific neutralizing antibodies (NA) exist in the majority of human immunodeficiency virus type 1 (HIV-1)-infected patients, whereas the NA response against autologous HIV-1 virus isolates is highly type-specific, motivated us to study the type- and group-specific NA responses generated upon presentation of escape virus, and the viral epitopes involved in the escape. Patients with demonstrable escape virus all developed group-specific NA, which were detectable after a delay and disappeared prior to disease development. The sera tested inhibited the binding of recombinant soluble gp120IIIB to cell-associated CD4, but group-specific virus neutralization required binding of NA to HIV-1 prior to viral attachment to target cells. Consecutive escape virus isolates were tested for sensitivity to neutralization by heterologous sera. Only minor differences were demonstrated, suggesting that the majority of the change in neutralization sensitivity is driven by the selective pressure of type-specific NA. Furthermore, no differences were observed in sensitivity to neutralization by anti-carbohydrate neutralizing monoclonal antibodies or the lectin concanavalin A, indicating a conserved nature of certain carbohydrate neutralization epitopes during escape. Finally the V3 sequence of three sets of consecutive virus isolates were analysed revealing amino acid mutations in V3 sequences of all escape virus isolates. The biological significance of these variations was confirmed further by the demonstration of changes in sensitivity to neutralization by anti-V3 monoclonal antibodies. These results strongly suggest a participation of the NA response against the V3 loop in the immunoselection of escape virus.

Immune response to immunostimulatory complexes (ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120)

In mice, immunostimulatory complexes (ISCOMs) prepared from HIV-1 B external envelope glycoprotein (gp120) induced 10-fold higher antibody titres than gp120 emulsified in depot adjuvant, as measured by enzyme-linked immunosorbent assay (ELISA). Rhesus monkeys immunized with gp120 ISCOMs produced precipitating and virus neutralizing antibody titres equivalent to those seen in HIV-infected chimpanzees and humans. After multiple immunizations with HIV-1 B gp120 ISCOMs, a rhesus monkey developed a neutralizing response to the HIV-1 isolates RF and MN, but not to the CC isolate. Antisera from ISCOM-immunized rhesus monkeys recognized gp120 on the membranes of HIV-1 B-infected H9 cells, indicating the preservation of epitope structure in the ISCOMs matrix.

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Adjuvanticity of ISCOMs incorporating a T cell-reactive lipoprotein of the facultative intracellular pathogen Francisella tularensis.

Immunostimulating complexes (ISCOMs) are known to be highly effective adjuvants for envelope antigens of viral agents, but have not been evaluated for use with antigens of intracellular bacteria. Balb/c mice were subcutaneously immunized with ISCOMs into which the T cell-reactive membrane protein TUL4 of Francisella tularensis had been incorporated. Spleen cells from the immunized mice responded in vitro to TUL4 and to heat-killed F. tularensis live vaccine strain (LVS) with proliferation and production of gamma-interferon, whereas spleen cells from control mice immunized with TUL4 only did not respond to the antigens. When mice immunized with TUL4 ISCOMs were challenged with F. tularensis LVS, bacterial counts in spleen and liver were lower than in non-immunized mice. Again, TUL4 had no effect when used without ISCOMs. When proteins of a total membrane preparation of F. tularensis LVS were incorporated in ISCOMs and used for immunization, a decrease in bacterial counts was obtained which was similar in magnitude to that of TUL4 ISCOMs. Generally, the adjuvant effects demonstrated did not compare with the excellent protective effect of live tularaemia vaccine. Nonetheless, ISCOMs provide a means whereby protective antigens of F. tularensis can be tested.

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4.

SummaryGlycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.