Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.     

Viral protein expression in producer and nonproducer clones of friend erythroleukemia cell lines.

Erythroleukemia cell lines HFL/d and HFL/b, derived from tumors induced in vivo in BALB/c (H-2d) and congenic BALB.B (H-2b) mice, respectively, by a polycythemia-inducing strain of Friend virus, produced both spleen focus-forming virus (SFFV) and its native NB-tropic helper virus (Friend murine leukemia virus [FMuLV]) during early-passage generations in culture. Eventually each line ceased production of both infectious viruses but retained its tumorigenic potential in syngeneic hosts. Virus-producer and -nonproducer clones of these cell lines were examined for expression of proteins encoded by the SFFV or FMuLV genomes. Lysates of labeled cells were treated with various antiviral sera, and the precipitates were examined by gel electrophoresis. Expression of the FMuLV env gene-encoded precursor protein, gPr84env, was observed in all producer and most nonproducer clones, but the FMuLV gag and pol gene products, Pr65gag and Pr200gag-pol, were uniformly undetectable in nonproducer clones. All HFL/d and HFL/b clones expressed appreciable amounts of the SFFV-encoded envelope protein, gp52, including one exceptional clone which had ceased to express any FMuLV-encoded proteins. The molecular weight of this SFFV-encoded envelope protein was consistently smaller in all HFL/b clones than in HFL/d clones, regardless of their producer or nonproducer status. The virus-nonproducer phenotype thus appears to be due to shutdown of expression of the 5' portion of the FMuLV genome in two independent cell lines.     

Plasma membrane glycoproteins encoded by cloned Rauscher and Friend spleen focus-forming viruses.

Rauscher spleen focus-forming virus (SFFV) was cloned free of its helper virus into normal rat kidney and mouse fibroblasts, and the resulting nonproducer fibroblast clones were analyzed. Our results suggested that Rauscher SFFV encodes a glycoprotein with an apparent Mr of 54,000 (gp54) that reacts with antisera made to the envelope glycoprotein (gp70) of ecotropic murine leukemia viruses, as well as with a rat antiserum that reacts with the gp70's of dual-tropic mink cell focus-inducing and HIX viruses but not with the gp70's of ecotropic viruses. In these respects and in its tryptic peptide map, Rauscher SFFV-encoded gp54 is nearly identical to the gp55 glycoprotein which we previously reported to be encoded by Friend SFFV (Dresler et al., J. Virol. 30:564--575, 1979). However, gp54 is slightly smaller, and it lacks one methionine-containing tryptic peptide that occurs in gp55. Studies with cytotoxic antiserum in the presence of complement and with a rosetting technique which employed sheep erythrocytes coupled to protein A suggested that the gp54 and gp55 glycoproteins are weakly expressed on the surface membranes of SFFV-infected cells. In addition, the Rauscher SFFV genome also encodes gag polyproteins which appear to be identical to the gag polyproteins encoded by helper Rauscher murine leukemia virus, but differ from the antigenically related polyproteins encoded by some but not all clones of Friend SFFV. Furthermore, the glycosylated gag polyproteins encoded by Rauscher SFFV and by some Friend SFFVs also appear to be expressed on the surface membranes of infected cells. These results suggest that similar env gene recombination and partial deletion events were involved in the independent origins of two different strains of acute erythroleukemia virus.     

Natural immunity in mice to the envelope glycoprotein of endogenous ecotropic type C viruses: neutralization of virus infectivity.

The ability of naturally immune mouse sera to neutralize ecotropic AKR murine leukemia virus (MuLV) was examined by using unfrozen virus preparations harvested for 1 h. In this assay several mouse sera significantly and consistently neutralized MuLV infectivity. The ability of these sera to neutralize was correlated with the presence of antibodies against MuLV detectable in a radioimmune precipitation assay using radioactively labeled intact virions. This neutralization was specific, in that either N- or B-tropic viruses, but not Friend MuLV, were neutralized. In addition, neutralization could be abrogated with purified AKR MuLV gp71 at concentrations that do not interfere with virus infectivity but could not be abrogated with Rauscher MuLV gp71. Neutralizing activity could be removed by absorption with intact AKR MuLV, but not by absorption with Friend MuLV, a BALB/c xenotropic virus, or with NZB xenotropic virus. All the neutralizing activity of (B6C3)F1 mouse sera was associated with the immunoglobulin G fraction.     

Loss of pathogenicity of spleen focus-forming virus after pseudotyping with Akv.

Friend virus complex (FV), which comprises replication-competent Friend murine leukemia virus (FMuLV) plus replication-defective spleen focus-forming virus (SFFV), induces a multistage erythroleukemia. We have examined the role of replication-competent helper virus in the early and late stages of FV disease by replacing FMuLV, the native helper, with Akv, the endogenous ecotropic MuLV of AKR mice. SFFVP/FRE, an established fibroblast line nonproductively infected with the polycythemic strain of SFFV, was superinfected with FMuLV or with Akv. Although supernatants from these cells showed similar titers in the XC plaque assay, supernatants from Akv-infected SFFVP/FRE cells showed 100- to 5,000-fold less activity than did those from FMuLV-infected cells with respect to spleen focus induction in vivo. Since virions isolated from these two supernatants contained similar ratios of SFFV to helper virus genomic RNA, it did not appear that the difference was due to a relative inability of Akv to package SFFV. Although FMuLV- and Akv-rescued SFFV are equally infectious in a mouse fibroblast cell line (NIH 3T3), FMuLV-rescued SFFV was far more efficient in inducing erythroid bursts in cultured primary bone marrow cells. Adding Akv to preparations of FMuLV-rescued SFFV did not significantly interfere with burst induction. Helper-free SFFV induced 50- to 500-fold more spleen foci when coinjected with FMuLV than it did with Akv. Helper virus also affected mortality rates that reflect the late stage of the disease. When FMuLV- or Akv-rescued SFFV was injected into NIH Swiss mice at dosage levels adjusted to give equal numbers of spleen foci, all mice receiving FMuLV-rescued SFFV developed splenomegaly and died, whereas no mice receiving Akv-rescued SFFV died or developed detectable splenomegaly. When FMuLV was coinjected with Akv-rescued SFFV, the mortality rate rose from 0 to 100%. Injection of helper-free SFFV alone did not induce mortality, but coinjection of helper-free SFFV with FMuLV resulted in 100% mortality. Thus, the helper virus used to rescue SFFV plays at least a quantitatively important role in the early stage of FV disease and a crucial role in the late stage of the disease in vivo.     

Helper-Dependent Properties of Friend Spleen Focus-Forming Virus: Effect of the Fv-1 Gene on the Late Stages in Virus Synthesis

Co-infection of neonatal BALB/c mice with Friend virus (FV) complex (containing defective spleen focus-forming virus [SFFV] and endogenous N-tropic leukemia-inducing helper virus [LLV-F]) and B-tropic Tennant leukemia virus (TenLV) resulted in the inhibition of LLV-F by the Fv-1(b) gene and recovery of a TenLV pseudotype of SFFV, abbreviated SFFV(TenLV). The host range of this pseudotype was B-tropic, since SFFV(TenLV) was 10 to 100 times more infectious for B-type (Fv-1(bb)) than for N-type (Fv-1(nn)) mice. The similar patterns of neutralization of N-tropic and B-tropic SFFV by type-specific murine antisera suggested that the difference in infectivity between these two SFFV preparations did not reside in envelope determinants. Rather, helper control of SFFV's host range was only apparent and dependent upon the ability of associated virus to provide a helper function for late stages in SFFV synthesis. Early stages in SFFV's infectious cycle were shown to be helper independent. The Fv-1 gene did not act at the level of the cell membrane to effectively restrict SFFV infection, since SFFV-induced transformed cells could be detected in the absence of spleen focus formation and SFFV synthesis. Further, the generation of these transformed cells by SFFV followed a one-hit, dose-response pattern, suggesting that SFFV-induced cell transformation is helper independent. Finally, restriction of helper function by Fv-1 may be an intracellular event, because both SFFV and its associated LLV-F helper share common envelope determinants and presumably adsorb onto and penetrate target cells with equal efficiency.     

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.     

Virus-specific phosphoproteins in simian sarcoma virus-transformed primate cells.

Cells transformed by simian sarcoma virus (SSV) express a 115000-dalton protein ( p115 ) that is precipitated by a goat antiserum to disrupted SSV/SSAV-infected and transformed cells but not by antibodies directed against the viral gag protein, p30, or envelope proteins. The protein is detected in productively as well as in nonproductively infected, transformed cells. It is not present in untransformed cells infected with helper virus (SSAV). The protein can be phosphorylated in vivo and in vitro at the tyrosine residue and SSV-transformed cells contain elevated levels of phosphotyrosine.     

Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates.

In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.     

Studies of the conformation-dependent neutralizing epitopes of simian immunodeficiency virus envelope protein.

It has been shown previously that the major neutralizing epitopes in simian immunodeficiency virus (SIV) are discontinuous and conformation dependent and that the V3 loop, in contrast to that of human immunodeficiency virus (HIV) type 1, does not by itself elicit neutralizing antibodies (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 89:1418-1422, 1992). We now present data showing that on the basis of fractionation of infected macaque sera, protease digestion of the envelope, and binding properties of two neutralizing monoclonal antibodies to SIV and SIV-HIV chimeric envelope proteins, changes in V3 can disrupt the conformation-dependent neutralization region. The chimeric protein did not produce significant neutralizing antibodies against either SIV or HIV. We also report that neutralizing antibodies elicited by recombinant SIV envelope proteins of mac251 and B670 isolates cross-neutralize. Finally, we show that deglycosylation of the SIV envelope results in a molecule which binds neither soluble CD4 nor the neutralizing monoclonal antibodies being investigated here and does not elicit sera with a significant neutralizing titer.     

Augmentation of helper virus replication in the presence of defective retrovirus

The interaction between defective spleen focus-forming virus (SFFV) and helper virus(es) in Friend virus (FV) complex has been assumed to be one-way, with the helper virus complementing SFFV by supplying necessary virion components. To test this assumption the expression of both SFFV and helper virus in partially congenic mice which differ at the Fv-2 locus, a gene that specifically controls susceptibility to SFFV, was analysed. When the mice were infected with LLV (a strain of Friend SFFV-free helper virus), there was no detectable effect of Fv-2 genotype on LLV expression as tested by virus infectivity in the XC plaque assay or by quantitative viral antigen analysis in an immunoprecipitation assay. However, after infection with FV complex there was an amplification of LLV (as well as SFFV) synthesis in Fv-2s as compared with Fv-2r hosts. To determine whether the increased LLV synthesis in Fv-2s mice was due to an increased population of susceptible target cells as a result of SFFV infection and/or transformation, the ratios of LLV-infected cells in the spleens of LLV- and FV-infected Fv-2s hosts in an infectious centre assay, were compared. Since the percentage of LLV-infected cells was equivalent in both instances, the higher rate of LLV synthesis after infection with FV complex was presumably due to intrinsic properties of SFFV-infected erythroid cells.     

Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity.     

Structural Proteins of Mammalian Oncogenic RNA Viruses: Murine Leukemia Virus Neutralization by Antisera Prepared Against Purified Envelope Glycoprotein

Goat and rabbit antisera prepared against a purified Rauscher murine leukemia virus glycoprotein (gp69/71) rapidly neutralized spleen focus-forming virus in Rauscher and Friend virus preparations. Absorption studies revealed that most of the neutralizing activity of goat anti-Rauscher virus gp69/71 serum was directed against type- and group-specific determinants.     

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

The Friend virus genome: Evidence for the stable association of MuLV sequences and sequences involved in erythroleukemic transformation

Studies on the genetics and molecular biology of the Friend virus complex, which includes both a spleen focus-forming virus (SFFV) and a lymphoid leukemia helper virus (LLV), have been hampered by the apparent inability to propagate SFFV in vitro under clonal conditions. The present study describes the establishment of an NIH/3T3 mouse fibroblast culture which continuously releases high titers of both LLV and SFFV into the culture medium. SFFV harvested from such cultures was in excess of its LLV helper virus and titrated in vivo with multi-hit kinetics. Hybridization experiments, using purified 70S viral RNA and cDNA made from Friend virus stocks containing SFFV in excess of its helper LLV, indicated that approximately 25–30% of this cDNA represents SFFV-specific sequences. By use of this virus stock, several mouse and rat clones nonproductively infected with SFFV were isolated. SFFV rescued from these nonproductively infected clones by superinfection with LLV, as well as SFFV produced by chronically infected NIH/3T3 cells, was subject to restriction by a previously described host regulatory gene, Fv-2. Each of several SFFV nonproducer clones was shown to contain relatively large amounts of viral-specific RNA sequences. Moreover, these clones also expressed high levels of a 15,000 molecular weight virion structural protein, p15, while the levels of the other gag gene-coded proteins and the major viral envelope glycoprotein, gp70, were similar to those exhibited by uninfected cells. The stable association between the erythroleukemic activity of SFFV and the gag gene-coded protein, p15, of murine leukemia virus is discussed in terms of a possible model for the generation of the SFFV genome.     

Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line

Erythroid cells from mice infected with the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP), unlike normal erythroid cells, can proliferate and differentiate in apparent absence of the erythroid hormone erythropoietin (Epo). The unique envelope glycoprotein encoded by SFFV has been shown to be responsible for this biological effect. The recent isolation of an Epo-dependent erythroleukemia cell line, HCD-57, derived from a mouse infected at birth with Friend murine leukemia virus, afforded us the opportunity to study the direct effect of SFFVP on a homogeneous population of factor-dependent cells. The introduction of SFFVP in complex with various helper viruses into these Epo-dependent cells efficiently and reproducibly gave rise to lines which expressed high levels of SFFV and were factor independent. SFFV appears to be unique in its ability to abrogate the factor dependence of Epo-dependent HCD-57 cells, since infection of these cells with retroviruses carrying a variety of different oncogenes had no effect. The induction of Epo independence by SFFV does not appear to involve a classical autocrine mechanism, since there is no evidence that the factor-independent cells synthesize or secrete Epo or depend on it for their growth. However, the SFFV-infected, factor-independent cells had significantly fewer receptors available for binding Epo than their factor-dependent counterparts had, raising the possibility that the induction of factor independence by the virus may be due to the interaction of an SFFV-encoded protein with the Epo receptor.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

Human immunodeficiency virus neutralizing antibodies recognize several conserved domains on the envelope glycoproteins.

Serum neutralizing antibodies against the human immunodeficiency virus were frequently detected in infected individuals, and low or absent serum neutralizing titers correlated with poor prognosis. Multiple diverse human immunodeficiency virus isolates were found to exhibit similar susceptibility to neutralization by a panel of human seropositive sera, suggesting that neutralizing antibodies are largely directed against conserved viral domains. Furthermore, utilizing antisera raised against a library of synthetic env peptides, four regions which are important in the neutralization process have been identified within both human immunodeficiency virus envelope glycoproteins (gp41 and gp120). Three of these are in conserved domains and should be considered for inclusion in a candidate vaccine.     

Analysis of translational products of Friend strain of spleen focus-forming virus.

We have analyzed normal rat kidney cells nonproductively infected with the Friend strain of spleen focus-forming virus (SFFV) for the presence of murine leukemia virus-specific type C viral proteins. SFFV was found to code for the p15 and p12 proteins of Friend-murine leukemia virus as determined by immunological typing of their antigenic determinants. Molecular-size analysis of p15 and p12 proteins in SFFV nonproductively infected normal rat kidney cells indicated that these proteins are translated as parts of polyprotein molecules. The apparent molecular weights of the polypeptides bearing p12 antigenic determinants revealed the presence of translational products of the SFFV genome that could not be accounted for by know type C virus helper structural proteins.     

Protection against Friend retrovirus-induced leukemia by recombinant vaccinia viruses expressing the gag gene.

High sequence variability in the envelope gene of human immunodeficiency virus has provoked interest in nonenvelope antigens as potential immunogens against retrovirus infection. However, the role of core protein antigens encoded by the gag gene in protective immunity against retroviruses is unclear. By using recombinant vaccinia viruses expressing the Friend murine leukemia helper virus (F-MuLV) gag gene, we could prime CD4+ T-helper cells and protectively immunize susceptible strains of mice against Friend retrovirus infection. Recovery from leukemic splenomegaly developed more slowly after immunization with vaccinia virus-F-MuLV gag than with vaccinia virus-F-MuLV env; however, genetic nonresponders to the envelope protein could be partially protected with Gag vaccines. Class switching of F-MuLV-neutralizing antibodies from immunoglobulin M to immunoglobulin G after challenge with Friend virus complex was facilitated in mice immunized with the Gag antigen. Sequential deletion of the gag gene revealed that the major protective epitope was located on the N-terminal hydrophobic protein p15.