We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-␤-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 g/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colonystimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4 ؉ -and CD8 ؉ -T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.

Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine.

We have used recombinant vaccinia viruses expressing full-length or truncated gag or env genes of SIVmac239 to investigate the requirements for assembly of SIV proteins. We observed that assembly of virus-like particles (VLPs) was found to be 3- to 5-fold higher with full-length Env than with the truncated forms, or than VLPs containing only Gag proteins, in primary monkey cells or various human cell lines. When cells expressing Env proteins in the absence of Gag were examined by immunoelectron microscopy, clusters of Env protein and membrane vesicles containing Env proteins were observed at cell surfaces. A low level of vesicles was released from cells expressing full-length Env, but about a 10-fold higher level was released in cells expressing a truncated form of Env [Env733(t)] in which the cytoplasmic domain is only 17 amino acids in length. Another truncated protein, Env718(t), with a short cytoplasmic tail of 3 aa, was also incorporated into VLPs at a 10-fold higher level than the full-length Env protein and was more efficiently released in vesicles. The mature SU and TM proteins were predominantly incorporated into VLPs with full-length Env, but both cleaved and uncleaved precursor proteins were present in VLPs with truncated Env as well as in Env and Env(t) vesicles. A more prominent layer of spikes was seen by electron microscopy in VLPs with truncated Env than in VLPs containing full-length Env. These results indicate that truncated Env proteins have the ability to self-associate on the cell surface and are assembled into a more closely packed array than full-length Env, which could explain the preferential incorporation of Env proteins with short cytoplasmic tails into virions.

The effects of two functional domains, the membrane-proximal YXX⌽ motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXX⌽ motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.The human immunodeficiency virus (HIV) envelope glycoprotein (Env) mediates virus entry into cells and is also a major target for both cellular and antibody responses (21, 28). It is synthesized as a precursor molecule, gp160, which is subsequently processed into the surface subunit (SU) gp120 and the transmembrane subunit (TM) gp41 by a cellular protease, and exists as a trimer of gp120-gp41 heterodimers on viral or cell membranes (18, 52). gp120 interacts with receptor and coreceptor molecules for HIV and mediates virus attachment to the cell, while gp41 causes subsequent fusion between viral and cell membranes for releasing viral core components into the cell during the initial infection process (15). The TM protein consists of three distinct domains: the extracellular domain, the transmembrane domain, and the cytoplasmic domain.The Env protein of HIV as well as other lentiviruses has a long cytoplasmic domain with over 150 amino acids, in comparison to those of other retroviruses, which are about 30 to 50 amino acids in length. Early studies have shown that the long cytoplasmic domain of HIV Env plays important roles in regulating Env protein function and virus infectivity (17,24,33,51) and have identified several structural features modulating these functions, such as modulating surface expression of the Env protein (3,4,6,27,32,45,53), targeting Env protein to specific membrane microdomains for assembly (14,34,35,44,55), and interacting with the viral matrix protein for incorporation of the Env protein into released virions (1,11,16,20,22,39,57), as well as interacting with other cellular proteins (3,29,38,40,53). Of particular interest, two distinct regions have been identified in the long cytoplasmic domain of the HIV Env protein and shown to regulate its surface expression, a membrane-proximal Tyr-ba...

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SIVmac239 and SIVmac1A11 are wild-type viruses encoding Env proteins with full-length or truncated cytoplasmic tails (CTs), respectively. A mutant designated SIVmac239T has a site-specific mutation which introduces a stop codon in the env gene resulting a truncated protein of similar length to SIVmac1A11 Env. To investigate the role of specific sequence differences in these Env proteins, we constructed SIV mutants encoding 1A11 or 239 Env proteins with reciprocal exchanges of the CT or exchanges of both the surface unit (SU) and CT sequences. A truncated CT in the context of the 1A11 SU subunit was found to significantly enhance replication in CEMx174 (human T-cell line) and rhesus PBMCs. However, similar Env CT truncation did not enhance replication of SIVmac239 in human or monkey cells. SIVmac1A11 with a full-length SIVmac239 CT did not replicate in human T-cell lines, but truncation of the CT by a stop codon resulted in replication. We also observed that these viruses differed significantly in sensitivity to neutralization by antibody. Taken together, the results indicated that the length of the CT domain as well as specific sequence differences in the SU domain affect viral replication capacity as well as sensitivity to neutralization.

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