Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.     

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.     

Induction of antibody responses that neutralize human T-cell leukemia virus type I infection in vitro and in vivo by peptide immunization.

In order to define neutralization regions on the envelope antigen of human T-cell leukemia virus type I (HTLV-I), we have generated a number of new anti-envelope gp46 monoclonal antibodies from rats and mice. Epitopes recognized by new monoclonal antibodies which could neutralize HTLV-I in syncytium and transformation inhibition assays were localized to sequences in gp46 from amino acids 186 to 193, 190 to 195, 191 to 195, 191 to 196, and 194 to 199. Ovalbumin-conjugated synthetic gp46 peptides containing these neutralization epitopes, pep190-199 (a synthetic gp46 peptide containing amino acids 190 to 199) and pep180-204, but not pep185-194 or pep194-203, could give rise to HTLV-I-neutralizing antibody responses in rabbits. These immune or nonimmune rabbits were then challenged with HTLV-I by intravenous inoculation with 5 x 10(7) live HTLV-I-producing ILT-8M2 cells. By a PCR assay, it was revealed that HTLV-I provirus was detected in peripheral blood lymphocytes from nonimmune and pep288-312-immunized rabbits, whereas the provirus was not detected in peripheral blood lymphocytes from pep190-199- and pep180-204-immunized rabbits over an extended period. These results suggest that the induction of anti-gp46 neutralizing antibody responses by immunization with synthetic peptides has the potential to protect animals against HTLV-I infection in vivo.     

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein.

In order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.     

Study of the antigenic structure of tick-borne encephalitis virus using synthetic peptides]

A number of peptides, fragments of the envelope protein E of the tick-borne encephalitis virus (Sofjin strain), were synthesized. Their binding to the polyclonal antiserum to protein E was studied. Rats were immunized with both the free peptides and their KLH-conjugates, and the resulting antisera were tested for their reactivity toward protein E and for their neutralizing activity toward the virus in cell culture. The only peptide corresponding to the 98-113 sequence of protein E was shown to be bound by the protein E antiserum in EIA. Two-fold immunization of rats with KLH-conjugates of the peptides corresponding to the 98-113, 130-143, and 394-403 sequences of protein E resulted in antipeptide antibodies capable of binding the native protein E, and the antibodies to the 98-113 and 394-403 peptides were capable of neutralizing the virus.     

Identification of functional regions in the human T-cell leukemia virus type I SU glycoprotein.

Single conservative and nonconservative amino acid substitutions were introduced into the gp45 external envelope protein (SU) of human T-cell leukemia virus type I (HTLV-I). The mutated amino acids were those identified as being conserved in HTLV-I, HTLV-II, and simian T-cell leukemia virus type I (but not in bovine leukemia virus). The mutated envelopes were tested for intracellular maturation and for function. Mutants with three major phenotypes could be defined: (i) 9 mutants with a wild-type phenotype, which included most of the conservative amino acid changes (five of seven) distributed throughout the SU protein; (ii) 8 mutants with affected intracellular maturation, 6 of which define a region in the central part of the SU protein essential for correct folding of the protein; and (iii) 13 mutants with normal intracellular maturation but impaired syncytium formation. These mutations likely affect the receptor binding step or postbinding events required for fusion. Five of these mutations are located between amino acids 75 and 101 of the SU protein, in the amino-terminal third of the molecule. The other mutations involve positions 170, 181, 195, 197, 208, 233, and 286, suggesting that two other domains, one central and one carboxy terminal, are involved in HTLV-I envelope functions.     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.     

Identification of an 80-kilodalton membrane glycoprotein important for human T-cell leukemia virus type I and type II syncytium formation and infection.

Human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II, respectively) infect certain sublines of the BJAB human B-cell line. We observed that the WH subline, but not the CC/84 subline, of BJAB cells were infectible by cell-free HTLV-I or HTLV-II and formed syncytia with cells infected by these retroviruses. This suggests that the BJAB-CC/84 cells possibly lack a membrane molecule(s) important for syncytium formation and infectibility. In order to identify this antigen, we generated polyclonal anti-BJAB-WH antisera which were adsorbed on BJAB-CC/84 cells. The adsorbed antisera bound only BJAB-WH and BJAB-CC/79 cells as demonstrated by complement-dependent cytotoxicity and flow cytometric assays. Furthermore, this adsorbed antisera bound several human T-cell clones, including SupT-1, as determined by flow cytometric assays. The adsorbed antiserum was monospecific as it immunoprecipitated only one 78- to 80-kDa protein from lysates of metabolically labeled BJAB-WH, BJAB-CC/79, and SupT-1, but not BJAB-CC/84, cells. The monospecific antisera detected a glycoprotein composed of a 64- to 66-kDa core protein containing tunicamycin-sensitive N-linked oligosaccharides. This membrane glycoprotein appears to be involved in HTLV-I- and HTLV-II-induced fusion and infection, as the monospecific antisera were capable of inhibiting both of these processes. The monospecific antisera diluted 1:50 and 1:90 inhibited 85 to 90% of syncytium formation induced in BJAB-WH, BJAB-CC/79, and SupT-1 cells cultured with HTLV-I- or HTLV-II-infected MT2, MoT, or FLW human T- or B-cell lines. At the same dilution, antisera inhibited 70 to 80% of infection of BJAB-WH cells by cell-free HTLV-I or HTLV-II. Thus, these studies indicate a role for a 78- to 80-kDa glycoprotein in HTLV-I or HTLV-II infection and syncytium formation.     

Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range.

Several epidemiologic and clinical studies suggest that patients coinfected with human immunodeficiency virus (HIV), the primary etiologic agent in AIDS, and other viruses, such as cytomegalovirus or human T-cell leukemia virus (HTLV), have a more severe clinical course than those infected with HIV alone. Cells infected with two viruses can, in some cases, give rise to phenotypically mixed virions with altered or broadened cell tropism and could therefore account for some of these findings. Such pseudotypes could alter the course of disease by infecting more tissues than are normally infected by HIV. We show here that HIV type 1 (HIV-1) efficiently incorporates the HTLV type I (HTLV-I) envelope glycoprotein and that both HIV-1 and HTLV-II accept other widely divergent envelope glycoproteins to form infectious pseudotype viruses whose cellular tropisms and relative abilities to be transmitted by cell-free virions or by cell contact are determined by the heterologous envelope. We also show that the mechanism by which virions incorporate heterologous envelope glycoproteins is independent of the presence of the homologous glycoprotein or heterologous gag proteins. These results may have important implications for the mechanism of HIV pathogenesis.     

Epitopes recognized by the neutralizing antibodies of an HIV-1-infected individual.

Sera from individuals infected by HIV-1 usually neutralize multiple viral isolates. To determine the extent to which these neutralizing antibodies recognize a principal neutralizing determinant in the V3 region of the envelope protein gp120 (amino acids 308-332), one broadly neutralizing serum was fractionated by affinity chromatography on immobilized peptide columns. Antibodies that neutralize one isolate (HTLV-IIIMN) were substantially but not completely absorbed by the peptide corresponding to a portion of its V3 determinant, whereas the antibodies that neutralize two other isolates (HTLV-IIIB and HTLV-IIIRF) were not absorbed by homologous peptides corresponding to their neutralizing determinants. Neutralizing antibodies also failed to be absorbed by full length envelope protein gp160 and by two other envelope peptides previously reported to be broadly neutralizing epitopes (amino acids 254-274 and 735-752). We conclude that the infected individual had raised a type-restricted neutralizing response targeted at a linear epitope in the V3 region, and that broad neutralization resulted from recognition of epitopes not yet identified.     

Polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120 T helper cell sites and B cell neutralization epitopes

In previous studies, we have used antisera raised to envelope (env)-gene-encoded synthetic peptides to identify a region of (HIV) glycoprotein (gp) 120 env protein designated SP10 that contains a type-specific neutralizing determinant. To develop a polyvalent, synthetic peptide inoculum that can evoke both neutralizing antibodies and T cell proliferative responses to more than one HIV isolate, synthetic peptides containing type-specific neutralizing determinants of gp120 from HIV isolates HTLV-IIIB (IIIB), HTLV-IIIMN (MN) and HTLV-IIIRF (RF) were coupled to a 16 amino acid T cell epitope (T1) of HIV-IIIB gp120 and used to immunize goats. Goat antisera to each T1-SP10 peptide derived from the SP10 region of gp120 of IIIB, MN, and RF neutralized HIV isolates IIIB, MN and RF in a type-specific manner. Moreover, peripheral blood T cells from immunized goats also proliferated in a type-specific manner to peptides derived from gp120 of IIIB, MN, and RF. When combined in a trivalent inoculum, T1-SP10 peptides from HIV-1 isolates IIIB, MN, and RF evoked a high titered neutralizing antibody response to isolates IIIB, MN, and RF in goats and as well induced immune T cells to undergo blast transformation in the presence of peptides derived from gp120 of all three HIV isolates. The T1 portion of the T1-SP10 construct was shown to induce a B cell antibody response against determinants within the T1 peptide in addition to inducing T cell proliferative responses in immune goat T cells. Moreover, the SP10 portion of the T1-SP10 constructs not only induced B cell antibody production but also induced type-specific T cell proliferative responses localized to the C-terminal variable sequences of the SP10 peptides. Finally, the T1-SP10 peptide construct induced memory T cell proliferative responses to native gp120 env protein. Thus, combinations of homologous SP10 region synthetic peptides containing type-specific neutralizing determinants and T cell epitopes of HIV gp120 may be useful in man to elicit high titered neutralizing B cell responses and, as well, T cell responses to more than one HIV isolate.     

Retroviral infections transmitted by blood transfusion

Modifications in donor screening and the introduction of laboratory testing of donated blood for anti-HIV-1 and anti-HTLV-I have resulted in a significant reduction in the risks of retroviral infections from blood transfusion. Presently, the American Red Cross detects an average of eight carriers of human immunodeficiency virus, type 1 (HIV-1) per 100,000 otherwise acceptable blood donors (0.008 percent), compared with an average of 35 per 100,000 (0.035 percent) when testing for HIV-1 antibodies began in 1985. Surveillance studies in the United States indicate a small likelihood that HIV-2 carriers will pass current screening procedures and be accepted as blood donors. Even if an HIV-2-infected person were to be accepted as a blood donor, there is a 42-92 percent likelihood that this person's blood would be detected as infective for HIV-2 and excluded because of serological cross-reactions that occur in the EIA for HIV-1 antibodies. During 1989, which was the first year that donated blood was routinely tested for antibodies to human T-lymphotropic virus, type I (HTLV-I) in the United States, approximately nine in 100,000 donors (0.009 percent) were confirmed positive for antibodies to HTLV-I, and their donated blood was excluded. Subsequent testing has revealed that a significant number of these persons whose sera was reactive by the HTLV-I EIA were, in fact, infected by HTLV-II. Epidemiological studies of human retroviral infections (HIV-1, HIV-2, HTLV-I, and HTLV-II) continue to provide important data and direction for improving criteria for qualifying blood donors.     

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

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

Use of a chimeric synthetic peptide from the core p19 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection

A chimeric synthetic peptide incorporating immunodominant epitope of the p19 gag protein (116-134) and the gp46 env protein (178-200) of HTLV-II virus, separated by two glycine residues, was synthesized by conventional solid-phase peptide synthesis. The antigenic activity of this peptide was evaluated by Ultramicro Enzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-II positive sera (n = 9), anti-HTLV-I/II positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 1),and anti-HTLV-I positive sera (n = 14), while specificity was evaluated with samples from healthy blood donors (n = 20). The efficacy of the chimeric peptide in solid-phase immunoassays was compared with the monomeric peptides. Data demonstrated that the chimeric peptide was the most reactive because it detected antibodies to virus efficiently. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of HTLV-II infection.     

Identification of a neutralization epitope on the envelope gp46 antigen of human T cell leukemia virus type I and induction of neutralizing antibody by peptide immunization.

We have generated a number of mAb against various epitopes on the external envelope glycoprotein, gp46, of human T cell leukemia virus type I (HTLV-I) from a WKA rat immunized with a recombinant vaccinia virus containing the HTLV-I env gene. Among these mAb, one group of mAb, represented by a mAb designated LAT-27, could neutralize the infectivity of HTLV-I, as determined by a HTLV-I-mediated cell fusion inhibition assay. LAT-27 also interfered with transformation of normal T lymphocytes by HTLV-I in vitro. An antibody-binding assay using overlapping synthetic oligopeptides showed that LAT-27 bound specifically to 10-mer peptides that contained the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). Antibodies from HTLV-I+ humans interfered with the binding of LAT-27 to gp46 Ag. Sera from rabbits immunized with a LAT-27-reactive peptide, 190-199, conjugated with OVA, but not sera from OVA-immunized rabbits, reacted with gp46 Ag and neutralized infectivity of HTLV-I. These results show that the HTLV-I neutralization epitope recognized by LAT-27 locates to the gp46 amino acids 191-196, and that immunization with a peptide containing the LAT-27 epitope can elicit an HTLV-I neutralizing antibody response.     

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus.

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.     

Complete nucleotide sequence of a highly divergent human T-cell leukemia (lymphotropic) virus type I (HTLV-I) variant from melanesia: genetic and phylogenetic relationship to HTLV-I strains from other geographical regions.

The high prevalences of antibodies against human T-cell leukemia (lymphotropic) virus type I (HTLV-I) reported for remote populations in Papua New Guinea and the Solomon Islands and for some aboriginal populations in Australia have been verified by virus isolation. Limited genetic analysis of the transmembrane portion (gp21) of the envelope gene of these viruses indicates the existence of highly divergent HTLV-I strains in Melanesia. Here, we report the complete nucleotide sequence of an HTLV-I isolate (designated HTLV-IMEL5) from the Solomon Islands. The overall nucleotide divergence of HTLV-IMEL5 from the prototype HTLV-IATK was approximately 8.5%. The degree of variability in the amino acid sequences of structural genes ranged between 3 and 11% and was higher (8.5 to 25%) for the regulatory (tax and rex) genes and the other genes encoded by the pX region. Since HTLV-IMEL5 was as distantly related to HTLV-II as to the other known HTLV-I strains, it could not have arisen from a reocmbinational event involving HTLV-II but rather might be an example of independent viral evolution in this remote population. These data provide important insights and raise new questions about the origin and global dissemination of HTLV-I.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.     

Diagnosis of infection with human T-lymphotropic virus type II (HTLV-II) in Norwegian HIV-infected individuals

Sera from 298 HIV-infected individuals from Southern Norway were examined for antibodies against HTLV. 30 sera (10.1%) were HTLV-II positive and 1(0.3%) HTLV-I positive. 25 of the HTLV-II infected subjects were intravenous drug abusers (IVDAs), giving a prevalence of HTLV-II infection of 24.5% in this group. Examination of blood samples by polymerase chain reaction followed by restriction enzyme analysis or sequencing confirmed the serological diagnosis. To evaluate current screening and verification HTLV tests, 44 sera were examined using a gelatin particle agglutination test, 5 different enzyme-linked immunoassays (ELISA) and 4 Western blots (WB). While earlier ELISAs and WBs were inadequate, a recent ELISA and WB including recombinant envelope glycoproteins from both viruses permitted serological diagnosis and distinction between HTLV-I and HTLV-II. Thus, HTLV-II now spreads among IVDAs in a North-European country. Health authorities in other countries should estimate the magnitude of the problem to decide upon measures to avoid transmission through blood transfusion.