Recent advances on the use of structural biology for the design of novel envelope immunogens of HIV-1.

Many efforts have been made in the worldwide quest for a prophylactic HIV vaccine to end the AIDS pandemic, but none has yet succeeded. The lessons learned have repeatedly informed us that the traditional or conventional approaches directly using the pathogens or subunits will not be sufficient for an effective HIV/AIDS vaccine. Recent advances in structure-based technology have shown some promise in the quest for a better immunogen in HIV vaccine development. According to the basic binding structural relationship of an antigen and an antibody, structure-based antigen design could bring some hope for the development of an effective vaccine against HIV.

Rational modifications of HIV-1 envelope glycoproteins for immunogen design.

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will likely require the elicitation of broadly neutralizing antibodies as well as cellular responses. The HIV exterior envelope glycoprotein trimers, gp120, and the transmembrane glycoprotein, gp41, mediate entry and are the sole viral targets for neutralizing antibodies. However, as subunit immunogens the envelope glycoproteins do not efficiently elicit antibodies capable of neutralizing the extremely diverse array of viruses circulating in the human population. The preponderance of data suggest that inefficient generation of broadly neutralizing antibodies is due to naturally evolved mechanisms of immune evasion inherent in the unmodified HIV envelope glycoproteins. Because the established modes of anti-viral vaccine development, live-attenuation and virus inactivation have not yet been successful for HIV, we and others have focused on subunit vaccine design. In this review, we describe current approaches of rational modification of the envelope glycoproteins based upon structure, antigenicity, biochemistry and biophysics to alter the properties of the envelope glycoproteins such that, as subunit immunogens, they now better elicit broadly neutralizing antibodies. The application of structure-assisted, rational subunit vaccine design may be a general paradigm for future efforts to develop vaccines against emerging human pathogens.

A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and C HIV-1 primary viruses.

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wild-type (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes.

Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.

A retroviral-derived immunosuppressive peptide activates mitogen-activated protein kinases.

The highly conserved region within the retroviral transmembrane envelope proteins has been implicated in a number of retrovirus-associated mechanisms of immunosuppression. CKS-17, a synthetic peptide representing the prototypic sequence of the immunosuppressive domain, has been found to suppress numerous immune functions, disregulate cytokines, and elevate intracellular cAMP. In this report we show that using a human monocytic cell line THP-1, CKS-17 activates mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 and 2 (ERK1/2). Kinetic studies show that CKS-17 induces an acute increase of ERK1/2 activity followed by a rapid decrease and then a second sustained increase of ERK1/2. CKS-17 also activates MAP kinase/ERK kinase (MEK) with a similar induction pattern. Mutant THP-1 cells isolated in our laboratory, in which CKS-17 exclusively fails to activate cAMP, did not show the transient decrease of CKS-17-induced ERK1/2 phosphorylation. Pretreatment of THP-1 cells or mutant THP-1 cells with cAMP analog or forskolin followed by treatment with CKS-17 showed no activation of MEK or ERK1/2. These results indicate that CKS-17 activates the MEK/ERK cascade and that there is a cross-talk between CKS-17-mediated MEK/ERK cascade and cAMP in that the MEK/ERK cascade is negatively regulated by cAMP. These data present a novel molecular mechanism(s) by this highly conserved retroviral immunosuppressive component.

Inhibition of cell-free human T-cell leukemia virus type 1 infection at a postbinding step by the synthetic peptide derived from an ectodomain of the gp21 transmembrane glycoprotein.

To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.

Influence of amino acid substitutions on antigenicity of immunodominant regions of the HTLV type I envelope surface gylcoprotein: a study using monoclonal antibodies raised against relevant peptides.

By the use of sera of human T cell leukemia virus type I (HTVL-I)-infected individuals it was shown that amino acid substitutions at positions 192 (proline to serine) and 250 (serine to proline) in major immunodominant regions (175-199 and 239-261) of the surface envelope glycoprotein (gp46) of the virus may influence the humoral response. Since human sera are polyclonal in nature, one cannot readily discriminate between an immunoglobulin-specific recognition and multiple bindings of diverse antibodies. To overcome this difficulty we generated murine monoclonal antibodies to synthetic peptides mimicking all or portions of these gp46 regions. The reactivity of some of these antibodies to synthetic peptides harboring (or not harboring) the preceding amino acid substitutions at position 192 or 250, to denatured gp46 by Western blotting, and to live (variously substituted) HTLV-I-infected cells, combined with blocking experiments with various peptides, allow us to conclude that the major epitopes (positions 183-191, 190-197, 190-199, and 246-252) in the two immunodominant regions may elicit different antibody responses according to their sequences. It is worth noting that in a reporter gene inhibition assay, it was found that a neutralizing monoclonal antibody (MF1), the epitope for which is located between residues 190 and 197, had a high level of activity when cells (2060) harboring a gp46 with proline at position 192 were used and had no activity toward cells (1010) with a serine at this position. Therefore our results establish that certain amino acid substitutions of gp46 may drastically affect the antigenicity of the molecule and the biological activity of the antibodies elicited.

Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

Inefficient assembly limits transport and cell surface expression of HLA-Cw4 molecules in C1R.

HLA-C antigens are expressed to the cell surface at roughly 10% the level of HLA-B or -A, and their serological definition remains persistently difficult. To characterize the factors limiting surface expression, the processes of assembly and intracellular transport of HLA-Cw4 molecules were investigated in the C1R cell line. When appropriate peptides were added to cultured cells or in cell lysates significant amounts of conformed HLA-C molecules that associate with beta 2-microglobulin (beta 2 m) are detected, but are indeed not sufficient to restore expression to the level observed for HLA-A or -B molecules. Furthermore, a precursor/product relationship exists between the free class I heavy chain and the mature conformation of HLA-Cw4 molecules. Thus, HLA-C assembly promotes the conversion of HC-10-reactive molecules (weakly-beta 2m-associated non-ligand associated free HC form) into the beta 2m-associated class I molecules recognized by W6/32. To further investigate the factors that regulate cell surface expression, intracellular transport of HLA-Cw4 was studied in pulse chase analysis. In contrast to some HLA-A and B, maturation of HLA-Cw4 heavy chains and their export to the medial and trans-Golgi compartments are quite inefficient. After 4 h of chase period, roughly half of the pulse-labeled HLA-Cw4 molecules have transited to the medial-Golgi and acquired complex oligosaccharides characteristic of mature form. In addition, treatment with gamma-interferon does not appear to improve maturation of HLA-Cw4 heavy chains, suggesting that increased supply of peptides does not influence intracellular transport. Moreover, only a small fraction in the pool of HLA-Cw4 molecules was subsequently transported through the trans-Golgi network, as indicated by their acquisition of sialic acids. Taken together these studies show that HLA-Cw4 molecules are inefficiently transported through the Golgi apparatus and presumably retained in the endoplasmic reticulum or cis-Golgi compartment.

Protection of rabbits against HTLV-II infection with a synthetic peptide corresponding to HTLV-II neutralization region.

Rabbit immune sera raised against synthetic peptides of the HTLV-II envelope gp46 region were examined for HTLV-II neutralization ability by HTLV-vesicular stomatitis virus (VSV) pseudotype assay and syncytium inhibition assay. HTLV-II neutralization activity was detected in the sera against HTLV-II Env gp46, 80-103 but not in those to HTLV-II Env gp46, 171-196. Three rabbits immunized with the synthetic peptide of HTLV-II Env gp46, 80-103 and three non-immunized rabbits were challenged with intravenous inoculation of an HTLV-II-producing human cell line (MOT, 1 x 10(7) cells). The non-immunized rabbits showed seroconversion for HTLV-II after 2 weeks and maintained persistent infection but the immunized rabbits were protected from HTLV-II infection. Nested or repeated polymerase chain reaction revealed the presence of HTLV-II provirus sequences in the non-immunized rabbits but not in the immunized rabbits. These results suggest that peptide vaccination with a synthetic peptide corresponding to the HTLV-II neutralization region is useful for preventing HTLV-II infection.

Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor.

The endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) of the human immunodeficiency virus type 1 (HIV-1) by a cellular protease is required for full activation of the virus. In this study, processing of gp160 was analyzed in vitro using the Kex2p endoprotease from the yeast Saccharomyces cerevisiae as a processing enzyme model. Endoproteolytic processing was examined using a synthetic peptide that mimics the cleavage site of HIV-1 glycoprotein, and a recombinant gp160 bearing the entire sequence of the env gene product, including the conserved cleavage site Arg508-Glu-Lys-Arg511. Coexpression in BHK-21 of Kex2p and gp160 by recombinant vaccinia viruses demonstrates that Kex2p can correctly process the HIV-1 glycoprotein to gp120 and gp41. Furthermore, recombinant gp160 and peptide were used as substrates and subjected to proteolysis with purified membranes from an S. cerevisiae strain overproducing the Kex2p endoprotease. Treatment of recombinant gp160, which has an apparent molecular mass of 127 kDa, with Kex2p and Western blot analysis showed that the precursor was cleaved into two products of about 101 and 34 kDa apparent molecular mass. Amino acid sequencing of the NH2-terminus of the 34-kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and Ala512. Recombinant gp160 mutated at the sequence coding for the potential cleavage site, and mature recombinant gp120, however, were not cleaved when treated with Kex2p. In summary, our results show that Kex2p cleaves both the HIV-1 envelope glycoprotein precursor and a synthetic peptide mimicking the cleavage site of HIV-1 gp160 at the dibasic site, suggesting functional analogy between yeast Kex2p and the cellular protease responsible for the maturation of HIV-1 envelope glycoproteins in infected human cells.

A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition.

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is approximately 85% helical. The high degree of stable secondary structure in a synthetic peptide of this size suggests self-association typical of a coiled coil or leucine zipper. In biological assays, the peptide efficiently blocked virus-mediated cell-cell fusion processes as well as infection of peripheral blood mononuclear cells by both prototypic and primary isolates of HIV-1. A single amino acid substitution in the peptide greatly destabilized its solution structure as measured by CD and abrogated its antiviral activity. An analogue containing a terminal cysteine was oxidized to form a dimer, and this modification lowered the dose required for antiviral effect from 5 to about 1 microgram/ml. These results suggest that both oligomerization and ordered structure are necessary for biological activity. They provide insights also into the role of this region in HIV infection and the potential for development of a new class of antiviral agents.