Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.     

A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101-106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.     

Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d

DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses. Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes. In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1.     

Elicitation of neutralizing antibodies by intranasal administration of recombinant vesicular stomatitis virus expressing human immunodeficiency virus type 1 gp120

Recombinant viral vectors are useful tools for AIDS vaccine development. However, expression of HIV-1 envelope genes using viral vectors has not been successful in the induction of potent neutralizing antibodies in vivo. We took advantage of the strong immunogenicity of vesicular stomatitis virus (VSV)-based vector and expressed HIV-1 HXB2 gp120 gene in the recombinant VSV. Our results showed that HIV-1 gp120 protein expressed by the recombinant VSV retained the native conformation of the protein to some degree and was recognized by two well-characterized broad anti-HIV-1 neutralizing monoclonal antibodies b12, 2G12. We further showed that only one time intranasal immunization with the recombinant VSV led to production of anti-HIV-1 anti-sera in mice. In addition, we found that the anti-sera had the ability to neutralize not only HXB2 envelope-pseudotyped HIV-1 viruses but also HIV-1 pseudotyped viruses with JRFL envelopes. These results suggest that HIV-1 gp120 expressed by the recombinant VSV, in combination with the route of intranasal administration, is an effective strategy to evaluate the immunogenicity of HIV-1 envelope protein and its variants in mice.     

Characterization of human immunodeficiency virus type 1 (HIV-1) envelope variation and neutralizing antibody responses during transmission of HIV-1 subtype B

We analyzed neutralization sensitivity and genetic variation of transmitted subtype B human immunodeficiency virus type 1 (HIV-1) in eight recently infected men who have sex with men and the virus from the six subjects who infected them. In contrast to reports of heterosexual transmission of subtype C HIV-1, in which the transmitted virus appears to be more neutralization sensitive, we demonstrate that in our study population, relatively few phenotypic changes in neutralization sensitivity or genotypic changes in envelope occurred during transmission of subtype B HIV-1. We suggest that limited genetic variation within the infecting host reduces the likelihood of selective transmission of neutralization-sensitive HIV.     

Newcastle disease virus expressing human immunodeficiency virus type 1 envelope glycoprotein induces strong mucosal and serum antibody responses in Guinea pigs

Human immunodeficiency virus type 1 (HIV-1) is transmitted mainly through mucosal sites. Optimum strategies to elicit both systemic and mucosal immunity are critical for the development of vaccines against HIV-1. We therefore sought to evaluate the induction of systemic and mucosal immune responses by the use of Newcastle disease virus (NDV) as a vaccine vector. We generated a recombinant NDV, designated rLaSota/gp160, expressing the gp160 envelope (Env) protein of HIV-1 from an added gene. The gp160 protein expressed by rLaSota/gp160 virus was detected on an infected cell surface and was incorporated into the NDV virion. Biochemical studies showed that gp160 present in infected cells and in the virion formed a higher-order oligomer that retained recognition by conformationally sensitive monoclonal antibodies. Expression of gp160 did not increase the virulence of recombinant NDV (rNDV) strain LaSota. Guinea pigs were administered rLaSota/gp160 via the intranasal (i.n.) or intramuscular (i.m.) route in different prime-boost combinations. Systemic and mucosal antibody responses specific to the HIV-1 envelope protein were assessed in serum and vaginal washes, respectively. Two or three immunizations via the i.n. or i.m. route induced a more potent systemic and mucosal immune response than a single immunization by either route. Priming by the i.n. route was more immunogenic than by the i.m. route, and the same was true for the boosts. Furthermore, immunization with rLaSota/gp160 by any route or combination of routes induced a Th1-type response, as reflected by the induction of stronger antigen-specific IgG2a than IgG1 antibody responses. Additionally, i.n. immunization elicited a stronger neutralizing serum antibody response to laboratory-adapted HIV-1 strain MN.3. These data illustrate that it is feasible to use NDV as a vaccine vector to elicit potent humoral and mucosal responses to the HIV-1 envelope protein.     

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.     

抗人类免疫缺陷病毒1型广谱中和抗体的研究进展

现行抗反转录病毒治疗药物的联合应用可有效抑制艾滋病进程并显著延长患者寿命,但由于人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)潜伏库的存在,艾滋病迄今尚无法治愈。近年发现抗HIV广谱中和抗体能有效降低患者体内病毒载量并延缓疾病进程,为研发艾滋病疫苗和治愈策略带来了曙光,尤其是序贯免疫策略的使用极大推进了广谱中和抗体的开发和应用进程。2018年,美国食品药品管理局(Food and Drug Administration,FDA)批准了第1个临床应用的广谱中性单克隆和抗体,无疑为抗HIV单克隆抗体药物的研发注入了一支强心剂。本文围绕近年来抗HIV广谱中和抗体的研究进展进行综述,探讨未来广谱中和抗体研发面临的挑战。     

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.     

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.     

Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines

The ability to elicit an immune response to a spectrum of human immunodeficiency virus type 1 (HIV-1) gene products from divergent strains is a desirable feature of an AIDS vaccine. In this study, we examined combinations of plasmids expressing multiple HIV-1 genes from different clades for their ability to elicit humoral and cellular immune responses in mice. Immunization with a modified Env, gp145DeltaCFI, in combination with a Gag-Pol-Nef fusion protein plasmid elicited similar CD4(+) and CD8(+) cellular responses to immunization with either vector alone. Further, when mice were immunized with a mixture of Env from three clades, A, B, and C, together with Gag-Pol-Nef, the overall potency and balance of CD4(+)- and CD8(+)-T-cell responses to all viral antigens were similar, with only minor differences noted. In addition, plasmid mixtures elicited antibody responses comparable to those from individual inoculations. These findings suggest that a multigene and multiclade vaccine, including components from A, B, and C Env and Gag-Pol-Nef, can broaden antiviral immune responses without immune interference. Such combinations of immunogens may help to address concerns about viral genetic diversity for a prospective HIV-1 vaccine.     

Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1

Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man₁₇-BSA and mannan, by -d-GlcNAc₄₇-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human ₁-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal₁₇-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc₄₇-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures.     

Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.     

Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (approximately 1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (approximately 40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.     

Immunogenicity of constrained monoclonal antibody A32-human immunodeficiency virus (HIV) Env gp120 complexes compared to that of recombinant HIV type 1 gp120 envelope glycoproteins

One strategy for the generation of broadly reactive neutralizing antibodies (NA) against human immunodeficiency virus type 1 (HIV-1) primary isolates is to use immunogens that have constrained HIV-1 envelope gp120 conformations reflective of triggered envelope on the surface of virions. A major change in gp120 following binding to CD4 is the enhanced exposure of the CCR5 binding site. One inducer of CCR5 binding site epitopes on gp120 is the human anti-gp120 monoclonal antibody, A32. We have made cross-linked A32-rgp120(89.6) and A32-rgp120(BaL) complexes and have compared their immunogenicities to those of uncomplexed recombinant gp120(BaL) (rgp120(BaL)) and rgp120(89.6). A32-rgp120(89.6) and A32-rgp120(BaL) complexes had stable induced CCR5 binding site expression compared to that of uncomplexed rgp120s. However, the A32-rgp120 complexes had similar capacities in guinea pigs for induction of NA against HIV-1 primary isolates versus that of rgp120 alone. A32-rgp120(89.6) induced antibodies that neutralized 6 out of 11 HIV-1 isolates, while rgp120(89.6) alone induced antibodies that neutralized 4 out of 11 HIV-1 isolates. A32-rgp120(BaL) complexes induced antibodies that neutralized 4 out of 14 HIV-1 isolates while, surprisingly, non-cross-linked rgp120(BaL) induced antibodies that neutralized 9 out of 14 (64%) HIV-1 isolates. Thus, stable enhanced expression of the coreceptor binding site on constrained gp120 is not sufficient for inducing broadly neutralizing anti-HIV-1 NA. Moreover, the ability of HIV-1 rgp120(BaL) to induce antibodies that neutralized approximately 60% of subtype B HIV-1 isolates warrants consideration of using HIV-1 BaL as a starting point for immunogen design for subtype B HIV-1 experimental immunogens.     

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization. In the four homosexual transmission couples, MAb neutralization sensitivity of HIV in recipients did not correlate with the MAb neutralization sensitivity of HIV from their source partners, whereas the neutralization sensitivity of donor and recipient viruses involved in parenteral transmission was more similar. For a fraction (11%) of the HIV-1 variants analyzed here, neutralization by 2G12 could not be predicted by the presence of N-linked glycosylation sites previously described to be involved in 2G12 binding. Resistance to 2F5 and 4E10 neutralization did also not correlate with mutations in the respective core epitopes. Overall, we observed that the neutralization resistance of recently transmitted subtype B HIV-1 variants was relatively high. Although 8 of 10 patients had viruses that were sensitive to neutralization by at least one of the four broadly neutralizing antibodies studied, 4 of 10 patients harbored at least one virus variant that seemed resistant to all four antibodies. Our results suggest that vaccine antigens that only elicit antibodies equivalent to b12, 2G12, 2F5, and 4E10 may not be sufficient to protect against all contemporary HIV-1 variants and that additional cross-neutralizing specificities need to be sought.     

Native and recombinant herpes simplex virus type 1 envelope proteins induce human immune T-lymphocyte responses.

The abilities of whole herpes simplex virus type 1 (HSV-1) antigen (HSV-ag) and purified HSV-1 native and recombinant envelope proteins to stimulate in vitro T-lymphocyte responses were compared in patients with recurrent herpes labialis. Immunochemically purified preparations of native glycoproteins B, C, and D (ngB, ngC, ngD) from cultured HSV-1 as well as expressed recombinant plasmid preparations of gD (rgD-1t, rgD-45K) elicited lymphocyte proliferation (LT) and production of gamma interferon (IFN-gamma) and interleukin-2 (IL-2) only in seropositive individuals. The IFN-gamma induced by rgD-1t correlated with the time to the next herpetic lesion in 19 volunteers followed to recurrence (r = 0.69, P less than 0.008), although the magnitude and frequency of LT and IFN-gamma responses were lower with either recombinant or native purified antigens than with the whole-virus antigen. Combinations of ngB plus ngD or ngB plus ngC plus ngD stimulated more IFN-gamma, equivalent to whole-virus-antigen responses. Recombinant-derived human IL-2 also specifically increased LT and IFN-gamma responses in antigen-driven cultures. ngD stimulated IL-2 and LT responses similar to those of whole-virus antigen and higher than those of ngC. HSV-ag and ngB induced significantly higher titers of total IFN than could be accounted for by IFN-gamma; this was not seen for the other antigens, which induced only IFN-gamma. HSV-ag-driven Leu 2a-, plastic-nonadherent blood cells, unlike whole peripheral blood mononuclear cells, showed evidence of an increase and then a decline in the frequency of HSV-responsive cells after a lesion recurrence. These studies suggest that HSV-1 envelope proteins are capable of stimulating an immune T-helper-cell response which is associated with the prevention of human herpes simplex lesion recurrence. Although the whole virus probably contains additional important antigens, increasing concentrations or combinations of certain purified glycoproteins or the addition of nonspecific enhancers of T-lymphocyte function can drive in vitro immune responses to the same level as the complete set of viral antigens.     

Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry.     

Conservation of human immunodeficiency virus type 1 gp120 inner-domain sequences in lentivirus and type A and B retrovirus envelope surface glycoproteins

We recently described a sequence similarity between the small ruminant lentivirus surface unit glycoprotein (SU) gp135 and the second conserved region (C2) of the primate lentivirus gp120 which indicates a structural similarity between gp135 and the inner proximal domain of the human immunodeficiency virus type 1 gp120 (I. Hötzel and W. P. Cheevers, Virus Res. 69:47–54, 2000). Here we found that the seven-amino-acid sequence of the gp120 strand β25 in the C5 region, which is also part of the inner proximal domain, was conserved in the SU of all lentiviruses in similar or identical positions relative to the carboxy terminus of SU. Sequences conforming to the gp135-gp120 consensus for β-strand 5 in the C2 region, which is antiparallel to β25, were then sought in the SU of other lentiviruses and retroviruses. Except for the feline immunodeficiency virus, sequences similar to the gp120-gp135 consensus for β5 and part of the preceding strand β4 were present in the SU of all lentiviruses. This motif was highly conserved among strains of each lentivirus and included a strictly conserved cysteine residue in β4. In addition, the β4/β5 consensus motif was also present in the conserved carboxy-terminal region of all type A and B retroviral envelope surface glycoproteins analyzed. Thus, the antiparallel β-strands 5 and 25 of gp120 form an SU surface highly conserved among the lentiviruses and at least partially conserved in the type A and B retroviral envelope glycoproteins.Lentiviruses are a group of strictly exogenous retroviruses that infect a range of mammalian hosts. One characteristic of this group of retroviruses is the rapid sequence divergence observed between virus strains as well as different lentiviruses, which resulted in the evolution of viruses with large differences in genome organization and sequence (20). Most of the sequence homology between highly divergent lentiviruses is present in the gag and pol gene products (8, 21). Sequence homology between the envelope glycoproteins of different lentiviruses has previously been shown to occur only in the ectodomain of the transmembrane subunit (TM) but not in the surface unit (SU) glycoprotein (3, 8, 21–23). Due to this apparent lack of sequence conservation in lentiviral SU, it has been unclear how the SU of different lentiviruses are structurally related to each other. To address this question, we recently compared SU sequences from the gp120 from primate lentiviruses and the gp135 of small ruminant lentiviruses and found a statistically significant sequence similarity between the second conserved region (C2) of gp120 and a 99-amino-acid region from gp135 (10). Analysis of this gp120-gp135 sequence similarity in the context of the gp120 structure revealed a partial structural similarity between gp120 and gp135.The human immunodeficiency virus type 1 (HIV-1) gp120 core bound to CD4 is composed of two major domains, the inner and outer domains, and a minidomain composed of four antiparallel β-strands, the bridging sheet (13). Sequences from the C2 region form most of the β-strands of a two-helix, two-strand bundle and a five-stranded β-sandwich in the inner domain as well as some β-strands of the outer domain of gp120 (13). Most of the similarity motifs between gp135 and the C2 region of gp120 coincide with sequences corresponding to β-strands 4 through 8 in the HIV-1 gp120 inner domain and β-strands 11 and 12 in the outer domain (10). Significantly, all four cysteines that form two disulfide bonds in the proximal region of the gp120 inner domain as well as the first cysteine of the gp120 V3 loop in β12 (13, 15) are conserved in gp135, indicating a partial similarity between the tertiary structures of gp120 and gp135 (10).The most conserved sequences between gp120 and gp135 correspond to strands β4 and β5 in the five-stranded β-sandwich structure of the proximal region of the inner proximal domain of HIV-1 gp120. Two additional β-strands in this five-stranded β-sandwich are derived from C1 and C5 sequences of HIV-1 gp120 (13). We hypothesized that C1 and C5 sequences, which are part of a structurally conserved SU inner proximal domain, should also be conserved between gp120 and gp135 and possibly in the SU of other lentiviruses. Here we show that two short motifs located in the gp120 C2 and C5 regions which are part of an antiparallel β-sheet in the gp120 inner proximal domain are conserved in the lentiviruses, indicating that a surface of the inner domain of HIV-1 gp120 is conserved in the SU of other lentiviruses. In addition, the C2 motif is also present in the envelope glycoproteins encoded by A-type endogenous retroviral elements and type B retroviruses (type A and B retroviruses), suggesting a local structural similarity between the SU of lentiviruses and type A and B retroviruses.

Sequence motif of the C5 region of HIV-1 gp120 is present in the SU of all lentiviruses.

As the sequences of three of the five β-strands of the gp120 inner proximal domain β-sandwich are conserved in gp135, we first tried to determine whether the gp120-gp135 sequence similarity extends to the other two β-strands which are part of this structure. One of these strands is β1, located in the C1 region of gp120 (13). Although the sequence of β1 is relatively well conserved among the primate lentiviruses, it is only 3 amino acids long, and a reliable assignation of similar sequences in gp135 could not be done. The other strand of this β-sandwich structure is the 7-amino-acid long β25. This strand is antiparallel to β5, which is the most conserved sequence between gp120 and gp135 (10, 13). Strand β25 is located about 20 amino acid residues upstream from the carboxy terminus of HIV-1 gp120 in the C5 region, and its sequence is highly conserved among strains of primate lentiviruses (sequence KYKVVKI in HIV-1_HXB2; residues conserved between HIV-1 strains are underlined) (12, 24). The last residue of this motif has been shown to be important for anchoring of gp120 on gp41 (9), suggesting that β25 is a functionally important structure of the inner proximal domain of gp120 likely to be conserved in other lentiviral glycoproteins. Sequences similar to the HIV-1 gp120 β25 motif (C5 motif) were visually sought in the gp135 carboxy-terminal region. A similar sequence was found in the caprine arthritis-encephalitis virus (CAEV) and visna virus gp135 between 33 and 34 amino acid residues upstream from the carboxy terminus of gp135 (Fig. ​(Fig.1B).1B). Similar to the C5 motif sequence of primate lentiviruses, the gp135 C5 motif is highly conserved in the gp135 of small ruminant lentiviruses (4, 27, 31, 35, 36). The sequence similarity also included the strictly conserved residue L483 of HIV-1 gp120 in the preceding α-helix 5, which is part of the two-helix, two-strand bundle of the inner domain. Flanking regions of gp120 and gp135 did not show any sequence similarity (not shown). Due to its short length, the significance of the conservation of the C5 motif in gp120 and gp135 was unclear. If this motif is indeed part of a structurally or functionally important domain of SU and not due only to chance, it should also be conserved in the SU of other lentiviruses. Therefore, to establish the relevance of this sequence similarity, we determined whether the C5 motif was also present in the carboxy terminus of the SU of other lentiviruses. Open in a separate windowFIG. 1Alignment of the C2 (A) and C5 (B) motifs of the SU from lentiviruses and type A and B retroviruses. Numbers at the right of the alignments indicate the position of the last residue of the motif from the initiation codon. Letters above the alignment indicate residue positions within each motif. Black backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses for each position of the motifs. Gray backgrounds represent identical amino acids or conservative variations between the lentiviruses and type A and B retroviruses (but which are nonconservative with the residues in black background) for each position. Numbers in parentheses indicate the number of amino acids between the last position of the C5 motif and the carboxy terminus of SU for each lentivirus. Thick lines indicate sequences which are part of HIV-1 gp120 strands β4, β5, and β25 and helix α5 (13). HIV-1 and HIV-2, human immunodeficiency virus types 1 (strain HXB2, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"K03455","term_id":"1906382"}}K03455) and 2 (strain ROD, {"type":"entrez-nucleotide","attrs":{"text":"X05291","term_id":"60146"}}X05291); CAEV, caprine arthritis-encephalitis virus ({"type":"entrez-nucleotide","attrs":{"text":"M33677","term_id":"323294"}}M33677); Visna, visna virus ({"type":"entrez-nucleotide","attrs":{"text":"M10608","term_id":"470313"}}M10608); JSRV, jaagsiekte sheep retrovirus ({"type":"entrez-nucleotide","attrs":{"text":"M80216","term_id":"331338"}}M80216); EIAV, equine infectious anemia virus (AF033820); FIV, feline immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M73965","term_id":"323946"}}M73965); BIV, bovine immunodeficiency virus ({"type":"entrez-nucleotide","attrs":{"text":"M32690","term_id":"210706"}}M32690); JDV, jembrana disease lentivirus ({"type":"entrez-nucleotide","attrs":{"text":"U21603","term_id":"733067"}}U21603); HERV-K, human endogenous retrovirus K, type 2 genome ({"type":"entrez-nucleotide","attrs":{"text":"X82272","term_id":"757871"}}X82272); MMTV, mouse mammary tumor virus ({"type":"entrez-nucleotide","attrs":{"text":"X01811","term_id":"61630"}}X01811); MIAE, mouse intracisternal A-type element ({"type":"entrez-nucleotide","attrs":{"text":"M73818","term_id":"194061"}}M73818).Sequences conforming to the C5 motif consensus were also found in the SU of the equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and the bovine jembrana disease lentivirus (JDV), 19 to 23 amino acid residues from the carboxy terminus of SU, the same relative position as the C5 motif from the carboxy terminus of gp120 in primate lentiviruses (Fig. ​(Fig.1B).1B). This sequence similarity was clear when considering the chemical similarities of amino acid side chains (Gln/Glu, Tyr/Trp, Lys/Arg/Gln, Val/Leu, or Val/Ile). A survey of lentiviral SU sequences present in GenBank revealed that the C5 motif was also highly conserved between EIAV, FIV, and BIV strains. For example, the C5 motif was found to be strictly conserved in 64 of 69 EIAV gp90 sequences in GenBank and is also stable during in vivo persistent infection (16, 39). However, the little variation that is observed between strains of a given lentivirus follows the same pattern as variation between different lentiviruses, suggesting a common constraint on sequence variation in different lentiviruses. For example, position h of the C5 motif of HIV-1 gp120 can be either of the conservative variations Lys/Arg or Gln/Glu, the same amino acids present at position h in other lentiviruses. Similarly, position h of the C5 motif of CAEV in different strains is either Lys or Arg (35), two of the residues allowed at position h in HIV-1 gp120. In addition, position b in the C5 motif of most FIV gp100 sequences in GenBank is the conservative variation Gln or Glu, the same amino acids present at position b of the C5 motif in EIAV and the small ruminant lentiviruses, respectively. Although the C5 motif is present in all lentiviruses, the flanking sequences were not consistently conserved except for a few amino acids in some pairwise alignments (not shown). Therefore, although conservation of the C5 motif may not be statistically significant in some SU pairwise alignments, the presence of this motif in the same position relative to the carboxy terminus of SU in all lentiviruses indicates that strand β25 of gp120 is an important structural or functional domain conserved in all lentiviruses.

Sequences similar to an HIV-1 gp120 C2 motif are present in the SU of most lentiviruses.

Using computer-assisted searches, we were previously unable to find in EIAV, BIV, or FIV the same extensive region of similarity that is observed between the C2 region of gp120 and gp135 (10). However, the presence of the C5 (β25) motif in all lentiviruses suggests that sequences similar to gp120 β5, which is antiparallel to β25 and conserved between gp120 and gp135, are also present in degenerate form in other lentiviruses. Visual examination of SU sequences from different lentiviruses revealed the presence of a similar motif (C2 motif) in EIAV, BIV, and JDV although not in FIV (Fig. ​(Fig.1A).1A). This 12-amino-acid C2 motif encompasses most of gp120 β-strands 4 and 5 and includes a strictly conserved cysteine residue in the β4 region. The C2 motif is highly conserved between strains of EIAV and BIV. In EIAV, the C2 motif is stable during persistent infection, with few conservative changes observed (16, 39). In addition, the C2 motif was found to be strictly conserved in 176 of 179 EIAV gp90 sequences present in GenBank, despite considerable sequence variation in other regions.Although some positions of the C2 motif were not absolutely conserved, we found a common pattern of variation between distantly related lentiviruses. For example, position f of the C2 motif is either Pro in EIAV, HIV-2, and simian immunodeficiency virus or aromatic (Tyr or Trp) in the small ruminant lentiviruses BIV and JDV. Also, position h can be either Phe or Tyr even in closely related lentiviruses (HIV-1/HIV-2, visna virus/CAEV, or BIV/JDV), and position l can be either Arg or Lys in the primate and small ruminant lentiviruses or Gln, which is a common conservative substitution for Arg and Lys, in EIAV, BIV, and JDV. Therefore, the C2 motifs of different lentiviruses appear to have a common constraint on sequence variation, suggesting a structural or functional similarity between the HIV-1 gp120 C2 domain and the SU of EIAV, BIV, and JDV.The other previously described gp120-gp135 conserved motifs outside the β4/β5 region could not be identified in the SU of other lentiviruses, including the sequence of gp120 β8, which has a cysteine forming a disulfide bond with the conserved β4 cysteine. Although the C2 motif was not present in FIV gp100, a similar motif was identified in a location upstream from the FIV gp100 V3 region (sequence SYCTDPLQIPLI, amino acids 318 to 329; conserved residues are underlined), in a similar relative position from gp100 V3 as the C2 motif from the V3 region of HIV-1 gp120. However, some of the highly conserved positions of the motif (positions g, h, and j) were not conserved in FIV gp100, and the significance of this FIV gp100 motif is unclear.

C2 motif is present in type A and B retroviral envelope surface glycoproteins.

The conservation of two short motifs in distant regions of SU that are located close to each other in the tertiary structure of HIV-1 gp120 suggests that this region represents a domain of SU that is of structural or functional importance. The TM ectodomains from lentiviruses and type B retroviruses have been shown to have some sequence similarity (19, 34, 38). Therefore, we asked whether sequence similarity between the Env of lentiviruses and type B retroviruses extends to the C2 and C5 motifs of SU.The type A and B retroviruses have some sequence homology in SU, and most of the sequence homology is located in the carboxy-terminal region of SU (18, 38). Visual examination of SU sequences from the human endogenous retrovirus K (18), mouse intracisternal A-type element (26), the exogenous/endogenous mouse mammary tumor virus (25), and the exogenous/endogenous type B/D jaagsiekte sheep retrovirus (JSRV) and the closely related ovine enzootic nasal tumor virus (which encode type B retroviral envelopes) (6, 38) revealed a sequence closely related to the C2 motif in their conserved carboxy-terminal region (Fig. ​(Fig.1A).1A). This sequence represents one of the most conserved sequences in the SU of this group of retroviruses and is also conserved among different strains or members of endogenous families (not shown). Some positions of the C2 motif, such as positions c, d, and g, are strictly or almost completely conserved between the lentiviruses and type A and B retroviruses. However, more informative than the sequence similarity between lentiviruses and type A and B retroviruses is the lack of distinction between the patterns of sequence variation for each position of the motif within and between retrovirus groups, even between closely related viruses. For example, position e of the C2 motif within both the lentiviruses and type A and B retroviruses can be either Pro or basic/Gln; the “dimorphic” position f encodes only Tyr/Trp or Pro (except in HIV-1); position h encodes either Phe or Tyr in all sequences; position i encodes either Ala or a hydrophobic residue in most sequences; position j encodes either Ile, Leu, or Phe in all sequences; position k encodes either Leu, Ile, or Val in all sequences; and position l is preferentially Lys, Arg, or Gln in the lentiviruses and JSRV. Most of these degenerate positions represent very conservative variations (positions a and h through l) or a restricted number of nonconservative variations (positions e and f, in the turn between β4 and β5). The sequence conservation and common pattern of variation between the C2 motifs of lentiviruses and type A and B retroviruses indicate a similar structural or functional constraint on sequence variation in the SU of these two groups of viruses.In contrast to the type A and B retroviruses, sequences similar to the C2 or C5 motifs could not be found in the SU of the Moloney murine leukemia virus, bovine leukemia virus, human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), Rous sarcoma virus, feline RD114 endogenous retrovirus, baboon endogenous retrovirus, feline leukemia virus type A, the Mason-Pfizer monkey retrovirus, or any spumaretrovirus even when using the Findpatterns program of the GCG package (7).Here we show that two short SU motifs are highly conserved in the lentiviruses and that one of these motifs is also conserved in the type A and B retroviruses. Many of the pairwise alignments were not statistically significant when tested by the Monte Carlo simulation of the Bestfit program of the GCG package and could therefore be attributed to chance. However, when all lentiviral sequences are included in the analysis and the multiple alignment is interpreted in the context of the X-ray structure of HIV-1 gp120, the conserved C2 and C5 motifs have a clear structural significance. The conservation of these motifs indicates that the region of the HIV-1 gp120 inner proximal domain centered on the antiparallel β-strands 5 and 25 forms a highly conserved lentiviral SU surface and suggests a possible structural similarity between the SU of lentiviruses and type A and B retroviruses in that domain. Although the C2 motif is too short to rule out convergent evolution between the SU of lentiviruses and type A and B retroviruses, their sequence similarity in TM (19, 34, 38) supports a common origin for most or the entire env genes of these two retroviral groups.The reason for the disagreement between the different degrees of sequence similarity in the SU of lentiviruses and the phylogenetic analyses of the pol gene products is unclear but probably reflects differences in evolutionary rates in different lentiviruses or recombination events (19, 20). Precedents for recombination events between env genes of closely or distantly related retroviruses, deduced from phylogenetic analyses, have been described. An exchange of env sequences probably occurred between HTLV-1 and HTLV-2 (19) and between a type C retrovirus closely related to the avian reticuloendotheliosis virus and a type B retrovirus which originated the type D retroviruses (19, 38).Modeling of the trimeric SU complex on the virion surface indicates that strands β5 and β25 form part of the most virion-proximal surface of the gp120 core (14, 37). While none of the residues of the C2 motif was directly tested for interactions with TM, at least one of the residues of β25 in the C5 region of HIV-1 gp120, I491, is important for stable SU-TM interactions (9). Therefore, the conserved lentiviral SU surface may represent a common structure among lentiviruses and possibly type A and B retroviruses for anchoring SU on TM in the envelope glycoprotein complex. It is interesting that the C5 motif region, which forms a β-strand in the CD4-bound gp120 core, is included in a computer-modeled pocket structure postulated to be important in SU-TM interactions (28), suggesting a structural basis for SU shedding upon receptor-induced conformational change.The sequence of the HIV-1 gp120 outer domain, shown as a cross-hatched box in Fig. ​Fig.2,2, is included entirely between the C2 and C5 motifs (13). Our previous sequence analysis indicates that the gp135s of small ruminant lentiviruses have a similar inner/outer domain organization: most strands of the inner domain β-sandwich as well as β12, located in the outer domain immediately upstream from the gp120 V3 loop, are conserved between gp135 and the gp120 of primate lentiviruses (10). The identification of a homologue of gp120 β25 in gp135 about 290 amino acid residues downstream from the C2 motif provides further support for a similar domain organization in the SU of primate and small ruminant lentiviruses. Consistent with this interpretation, the putative outer domain of gp135, located between the C2 and C5 motifs, is highly glycosylated and contains more than 80% of the potential N-linked glycosylation sites of gp135 (11), similar to the heavy glycosylation of the gp120 outer domain (37). In this gp135 domain model, the distance between the C2 and C5 motifs in the primary structure of SU would indicate a larger relative size of the putative outer domain of gp135 than gp120 outer domain. The presence of the C2 and C5 motifs in EIAV, BIV, and JDV would also suggest an analogous inner/outer domain organization for the SU of these lentiviruses. However, the shorter sequence between the C2 and C5 motifs in EIAV, BIV, and JDV may indicate either a much smaller or absent outer domain in the SU of these viruses (Fig. ​(Fig.2).2). The conserved C2 motif of EIAV gp90 was shown to be part of a minor neutralization epitope recognized by a murine monoclonal antibody (1), suggesting that the EIAV C2 motif is better exposed on the virion surface than the C2 motif of gp120, compatible with a smaller or absent outer domain in gp90. Interestingly, the C2 motif of type A and B retroviruses is located in the carboxy terminus of SU (Fig. ​(Fig.2)2) and C5 appears to be absent, indicating that the surface glycoproteins of type A and B retroviruses, although possibly structurally related to the SU of lentiviruses, probably lack an outer domain homologue and have a different domain organization than the SU of lentiviruses. Open in a separate windowFIG. 2Location of the C2 and C5 motifs in retrovirus envelope glycoproteins. The Env glycoproteins (excluding the amino-terminal leader peptide) are drawn to scale and aligned by the SU-TM cleavage sites conserved in all retroviruses (dotted line). The SU and TM domains of Env are indicated by double arrows. The boundaries of the C2, V3, and C5 regions of HIV-1 gp120 are indicated by thick lines above the alignment, and the location of the HIV-1 gp120 outer domain sequence is shown by a cross-hatched box. The black and gray boxes in the SU domain indicate the positions of the C2 and C5 motifs, respectively. Asterisks represent the described PNDs of EIAV (1), visna virus (29), FIV (17), and T-cell-adapted strains of HIV-1 (33) and HIV-2 (2).The two conserved colinear motifs of lentivirus SU could be useful as structural points of reference for comparative structural studies of SU from different lentiviruses. Variable domains of SU are important in the mechanisms of host cell invasion, tropism determination, and immune evasion. In HIV-1, the third variable loop V3 of gp120 is the main target of neutralizing antibodies in tissue culture-adapted strains and also determines coreceptor usage and tropism (5, 30, 32, 33). Whether sequences in variable regions of SU in other lentiviruses that are functionally equivalent to the gp120 V3 loop are also structurally related to the gp120 V3 loop is not clear. The position of variable domains relative to the C2 and C5 motifs could therefore indicate their structural relationship. For example, the principal neutralization domain (PND) of EIAV gp90, postulated to be functionally equivalent to the gp120 V3 loop (1, 16), is located upstream from the C2 motif instead of downstream, as the V3 loop in gp120 is (Fig. ​(Fig.2),2), suggesting that the gp90 PND and the gp120 V3 loop, while having similar roles in evasion of humoral immune responses, may not be structurally related to each other. A similar situation also occurs in visna virus, whose PND, located in the carboxy-terminal region of gp135 (29), was previously shown to be structurally unrelated to the HIV-1 gp120 V3 loop (10). This would indicate that different lentiviruses may have evolved different regions of a primordial lentivirus surface glycoprotein to perform similar functions important in virus-host interactions.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.