Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.

Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.     

DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation.

The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus.     

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus.

An E1-deleted, replication-defective adenovirus recombinant of the human strain 5 expressing the rabies virus glycoprotein, termed Adrab.gp, was tested in young mice. Mice immunized at birth with the Adrab.gp construct developed antibodies to rabies virus and cytokine-secreting lymphocytes and were protected against subsequent challenge. Maternal immunity to rabies virus strongly interferes with vaccination of the offspring with a traditional inactivated rabies virus vaccine. The immune response to the rabies virus glycoprotein, as presented by the Adrab.gp vaccine, on the other hand, was not impaired by maternal immunity. Even neonatal immunization of mice born to rabies virus-immune dams with Adrab.gp construct resulted in a long-lasting protective immune response to rabies virus, suggesting that this type of vaccine could be useful for immunization shortly after birth. Nevertheless, pups born to Adrab.gp virus-immune dams showed an impaired immune response to the rabies virus glycoprotein upon vaccination with the Adrab.gp virus, indicating that maternal immunity to the vaccine carrier affected the offspring's immune response to rabies virus.     

Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.

Herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) infect different natural hosts but are very similar in structure, replicative cycle, and entry into cultured cells. We determined whether HSV-1 and PRV use the same cellular components during entry into Vero cells, which are highly susceptible to each virus but are not from native hosts for either. UV-inactivated virions of either HSV-1 or PRV could saturate cell surfaces to block infection of challenge HSV-1 or PRV. In the presence of saturating levels for infection of either virus, radiolabeled virus bound well and in a heparin-sensitive manner. This result shows that heparan sulfate proteoglycans on Vero cells are not the limiting cellular component. To identify the virus component required for blocking, we used an HSV-1 null mutant virus lacking gB, gD, or gH as blocking virus. Virions lacking gB were able to block infection of challenge virus to the same level as did virus containing gB. In contrast, virions lacking gD lost all and most of the ability to block infection of HSV-1 and PRV, respectively. HSV-1 lacking gH and PRV lacking gp50 also were less competent in blocking infection of challenge virus. We conclude that HSV-1 and PRV bind to a common receptor for infection of Vero cells. Although both viruses bind a heparin-like cell component on many cells, including Vero cells, they also attach to a different and limited cell surface component that is bound at least by HSV-1 gD and possibly gH and to some degree by PRV gp50 but not gB. These results clearly demonstrate binding of both HSV-1 and PRV to a common cell receptor that is not heparan sulfate and demonstrate that several types of attachment occur for both viruses during infectious entry.     

Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.     

Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins.

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.     

Recombinant Sendai virus expressing the G glycoprotein of respiratory syncytial virus (RSV) elicits immune protection against RSV

Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.     

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.

Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.     

Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- PrV-infected animals exhibited severe symptoms, whereas the gp50- PrV-infected pigs showed a significant level of protection. In conclusion, vaccination with a PrV mutant lacking glycoprotein gp50, which is unable to spread between animals because of a lack of formation of free infectious virions, can confer on pigs protection against challenge infection. These results provide the basis for the development of new, nonspreading live herpesvirus vaccines based on gp50- PrV mutants.     

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.     

Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis.

We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.     

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.     

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.