Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production.

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV.

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery.

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine.

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiological studies of equine herpesvirus 1 (EHV-1) in Thoroughbred foals: a review of studies conducted in the Hunter Valley of New South Wales between 1995 and 1997.

Sero-epidemiological studies conducted between 1995 and 1997 on two large Thoroughbred stud farms in the Hunter Valley of NSW showed clear evidence of EHV-1 infection in foals as young as 30 days of age. Similarly, serological evidence suggested that these foals were infected with EHV-1 from their dams or from other lactating mares in the group, with subsequent foal to foal spread of infection prior to weaning. These studies also provided evidence of EHV-1 infection of foals at and subsequent to weaning, with foal to foal spread of EHV-1 amongst the weanlings. These data indicated that the mare and foal population was a reservoir of EHV-1, from which new cases of infection propagated through the foal population both before and after weaning. The results of these studies support the long standing management practices of separating pregnant mares from other groups of horses to reduce the incidence of EHV-1 abortion. Also, these results have important implications for currently recommended vaccination regimens, as the efficacy of vaccination in already latently infected horses is unknown.

Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a Hunter Valley stud farm: are mares the source of EHV-1 for unweaned foals.

The prevalence of EHV-1 and EHV-4 antibody-positive horses was determined using a type specific ELISA on serum samples collected from 229 mares and their foals resident on a large Thoroughbred stud farm in the Hunter Valley of New South Wales in February 1995. More than 99% of all mares and foals tested were EHV-4 antibody positive, while the prevalence of EHV-1 antibody positive mares and foals were 26.2 and 11.4%, respectively. Examination of the ELISA absorbance data for the individual mares and foals suggested that the EHV-1 antibody positive foals had been infected recently with EHV-1 and that a sub-group of the mare population was the likely source of infectious virus for the unweaned foals.

Toxicokinetics of Ro 5-4864, lindane and picrotoxin compared.

The effects produced by IP administration of these three agents in the rat were compared because of in vitro evidence that each modulates the picrotoxinin site of the GABAA receptor. For each, hypothermia had the lowest threshold and convulsions the next, with hypophagia produced only by the highest dose of either Ro 5-4864 or lindane. Convulsant effects had a shorter latency and a shorter duration than did hypothermia. Hypophagia, when present, lasted the longest. Myoclonus was the seizure type with the lowest threshold for all three agents. At the highest dose, lindane produced a high incidence of maximal clonic (hopping) seizures, whereas Ro 5-4864 and picrotoxin produced a high incidence of maximal tonic seizures instead. On a mole/kg basis, picrotoxin was 40 times more effective than the other two agents and produced seizures which started later, peaked later, and persisted longest. Ro 5-4864 and lindane were effective at equimolar concentrations and, in combination, produced effects which suggested either dose-addition or synergism. The data are consistent with the hypothesis that the toxic effects of both Ro 5-4864 and lindane may be attributable, at least in part, to an action at a subpopulation of GABAA receptors.

Application of an equine herpesvirus 1 (EHV1) type-specific ELISA to the management of an outbreak of EHV1 abortion.

Sera from 33 Australian thoroughbred mares were tested during an outbreak of equine herpesvirus 1 (EHV1) abortion with an enzyme-linked immunosorbant assay (ELISA) for the presence of EHV1-specific antibodies. The ELISA used a recombinant EHV1 antigen derived from glycoprotein G (gG) and distinguished antibodies to EHV1 from those of the antigenically related and widespread herpesvirus EHV4. Sera were obtained from most of the mares on three occasions, three, 13 and 67 days after the first abortion. Mares which were negative in the ELISA were kept separate from mares which were positive. A second abortion occurred two days after the first and two more abortions and one perinatal death occurred later. Sera from these last three mares showed a significant increase in EHV1-specific antibody on day 13 indicating a recent infection with EHV1. Ten other mares did not have antibodies to EHV1 on day 13 but had seroconverted to EHV1 by day 67. Despite the EHV1 infection, these mares foaled normally, possibly because the infection had occurred either late in gestation or after foaling. Seven mares that remained negative in the ELISA throughout the testing period did not abort, and neither did 11 mares that were positive in the ELISA when they were first tested.

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction.

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.

Seroprevalence of equine herpesvirus 1 in thoroughbred foals before and after weaning.

OBJECTIVE: To investigate the seroprevalence of equine herpesvirus 1 in foals around weaning and after weaning on two large Thoroughbred farms using a type-specific enzyme-linked immunosorbent assay to determine exposure to infection. DESIGN: A longitudinal population study in groups of Thoroughbred weanling foals. STUDY POPULATION: Two hundred weanling Thoroughbred foals from a population of about 380 foals were enrolled on two adjacent stud farms in the Hunter Valley of New South Wales. Foals on both farms were weaned from February to May 1995 into randomly selected groups of 10 to 15 foals. Farms were selected because of their willingness to cooperate in the survey and because their detailed records of foals and their movements. They were representative of well-managed large Thoroughbred stud farms in New South Wales. Both studs had upper respiratory tract disease among weanling foals around weaning each year although the seroprevalence of viral respiratory disease on either farm was not known before the study. PROCEDURE: Serum was collected from foals within each group at fortnightly intervals from 9th February until 1st June 1995, and at a single follow-up period in August 1995. Each sample was tested in triplicate using an antibody-detection ELISA which is type-specific for EHV-1 and EHV-4 antibodies. RESULTS AND CONCLUSIONS: There was serological evidence of EHV-1 infection both before and after weaning. The prevalence of EHV-1 antibody in the sample population increased during the study and individual cases of EHV-1 infection were identified. The increase was caused both by the seroconversion of foals within the groups and by the recruitment into the study of foals with pre-existing EHV-1 antibody. Evidence of EHV-1 infection in Thoroughbred foals after weaning has not been reported previously in Australia and this has implications for vaccination regimens.

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus.

A recombinant baculovirus, RecBV-E, encoding the hepatitis C virus (HCV) envelope proteins, E1 and E2, controlled by the cytomegalovirus promoter was constructed. RecBVs can infect mammalian cells, but fail to express proteins or replicate because the viral DNA promoters are not recognised. The RecBV-E transduced 86% of Huh7 cells and 22% of primary marmoset hepatocytes compared with 35% and 0.4%, respectively, after DNA transfection. Several stable cell lines were generated that constitutively expressed E1/E2 in every cell. No evidence of E1/E2-related apoptosis was noted, and the doubling times of cells were similar to that of the parental cells. A proportion of the E1/E2 was expressed on the surface of the stable cells as determined by flow cytometry and was detected by a conformation-dependent monoclonal antibody. It is likely that the continued expression of E1/E2 in the stable cells resulted from integration of the RecBV DNA. Infection of Huh7 cells, in the absence of G418 selection, failed to result in expression of the foreign gene (in this case, eGFP) beyond 14-18 days. RecBVs that express HCV genes from a CMV promoter represent an effective means by which to transduce primary hepatocytes for expression and replication studies.

Modulation of CD4+ T-cell recognition of influenza hemagglutinin by carbohydrate side chains located outside a T-cell determinant.

Two distinct T-cell clones raised to the H3 subtype influenza virus A/Memphis/71 fail to proliferate in response to other H3 viruses that contain the amino acid substitution Asp63-->Asn within the heavy (HA1) chain of the hemagglutinin (HA). These nonstimulatory viruses nevertheless have an intact sequence corresponding to the determinants recognized by the clones. The substitution at residue 63 results in the creation of a potential glycosylation site which occurs outside the boundaries of the T-cell determinants. A synthetic peptide encompassing the two determinants and extended to include residue Asn63 stimulated levels of proliferation of the clones similar to those obtained with a peptide containing Asp63. These results indicate that the presence of Asn rather than Asp does not in itself affect T-cell recognition. Partial enzymatic deglycosylation of isolated HA possessing the glycosylation site restored the ability of the T-cell clones to proliferate. More extensive deglycosylation of HA1 had a differential effect on the clones, allowing one to proliferate but not the other. These results illustrate that even when attached to residues outside a determinant, the carbohydrate of a glycoprotein antigen can influence T-cell recognition. In this way, changes in glycosylation patterns of HA from different isolates of the virus may influence the efficacy of preexisting T-cell immunity.

Conserved determinants for CD4+ T cells within the light chain of the H3 hemagglutinin molecule of influenza virus.

Helper T-cell clones were isolated from BALB/c mice that had been inoculated with purified light chain (HA2) from H3 subtype influenza virus hemagglutinin (HA). The clones were divided into two distinct groups based on their ability to proliferate in response to bromelain-derived HA (BHA) and the light chain derived from it (BHA2), both of which lack the C-terminal 46 amino acid residues of the HA2 chain. The first group contained two I-Ad restricted clones that proliferated in response to BHA and BHA2 and were found to recognize the determinant 96AELLVALEN104. The remaining seven clones were I-Ed restricted, required intact HA2 for proliferation, and responded to synthetic peptides containing the sequence 170RFQIKGVEL178 which spans the bromelain cleavage site. Although all T-cell clones proliferated in response to a wide range of different H3 virus strains, they showed no cross-reactivity with viruses of the H1 or H2 subtype. The T-cell clones from each group were able to provide help to virus-primed B cells allowing them to produce anti-HA antibody in vitro.

Equine gammaherpesvirus 2 (EHV2) is latent in B lymphocytes.

Peripheral blood leukocytes were collected from 5 Thoroughbred horses and examined for the presence of EHV2 in sub-populations of mononuclear cells. Peripheral blood mononuclear cells were separated on Percoll gradients and then enriched for plastic adherent cells (predominantly monocytes), surface immunoglobulin positive (sIg+) B lymphocytes and T lymphocytes, using panning techniques. The purity of each cell population was assessed by fluorescence activated cell scanning. In an infectious centre assay, each cell population was inoculated onto equine foetal kidney monolayer cell cultures which are fully permissive for the replication of EHV2. Only enrichment for sIg+ B lymphocytes resulted in a marked increase in the number of infectious centres, indicating that EHV2 is present in B lymphocytes. Freeze-thawing of sIg+ B lymphocytes, prior to inoculation onto EFK monolayer cell cultures, resulted in the complete abrogation of infectious centre formation, confirming that EHV2 is latent in B lymphocytes i.e., infectious free virus was not present in the cells. The number of EHV2 infected B lymphocytes varied considerably between horses from 4 to 780 per 10(6) cells. Evidence was also obtained that direct cell to cell contact between the epithelial cells and sIg+ B lymphocytes was necessary for the production of infectious centres. The data indicate that EHV2, like other members of the Gammaherpesvirinae, is latent within B lymphocytes.

The nucleotide sequence of the glycoprotein G homologue of equine herpesvirus 3 (EHV3) indicates EHV3 is a distinct equid alphaherpesvirus.

EHV3 causes equine coital exanthema and has been classified as an alphaherpesvirus on the basis of its biological properties; however due to the absence of any sequence information the phylogenetic relationship has not previously been examined. The complete nucleotide sequence of the EHV3 glycoprotein G (gG) gene was determined and showed that this virus is most closely related to the alphaherpesviruses equine herpesviruses type 1 (EHV 1) and type 4 (EHV4). EHV3 gG contains conserved and variable regions which are homologous to those previously defined for EHV1 and EHV4 gG proteins. Consistent with EHV1 and EHV4 gG, the variable region of EHV3 gG was found to elicit a strong antibody response in experimentally and naturally infected horses and could be exploited for use as a diagnostic reagent.

Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2.

Twenty-two monoclonal antibodies (MAbs) were generated to the gammaherpesvirus equine herpesvirus-2 (EHV-2). Using Western blot analysis, eight MAbs recognized an Escherichia coli glutathione S-transferase (GST)-glycoprotein B (gB) fusion protein and, using overlapping GST-gB fusion proteins, a neutralization epitope was mapped to amino acids 29-74. One of the gB-specific MAbs was used to characterize the glycosylation and kinetics of synthesis of EHV-2 gB. EHV-2 gB is synthesized as a 97 kDa polypeptide that is co-translationally modified to a 130 kDa high-mannose precursor that forms a 260 kDa dimer shortly after synthesis. Each 130 kDa precursor is endoproteolytically cleaved to disulphide-linked subunits of 75 and 58 kDa prior to further processing to complex oligosaccharide-containing subunits of 89 and 65/62 kDa. The 89 and 65/62 kDa subunits of EHV-2 gB contain 39 and 17 kDa of N-linked oligosaccharides, respectively, and do not contain any O-linked oligosaccharides. Western blot analysis of purified EHV-2 virions established that gB exists as a 320 kDa dimer in the virion envelope.

Equine herpesvirus-4 glycoprotein G is secreted as a disulphide-linked homodimer and is present as two homodimeric species in the virion.

Glycoprotein G (gG) homologues have been found in most alphaherpesviruses although little is known about their structure or function. In this study, three species of equine herpesvirus-4 (EHV-4) gG were identified: a full-length 68 kDa virion-associated species (gGVL), a 12 kDa virion-associated species (gGVS) and a 60 kDa secreted species (gGS), detected in the medium of infected cells. gGS and gGVS appear to be proteolytic cleavage products of gGVL and correspond to the N- and C-terminal regions, respectively. It was shown that gGS and gGVL are similarly glycosylated possessing mostly N-linked complex-type carbohydrate side chains. Western blots of proteins separated under nonreducing conditions established that gGS is secreted as a 120 kDa glycoprotein while the virion-associated species, gGVL and gGVS, are present in the virion as 140 and 20 kDa proteins, respectively. As gGS and gGVL do not appear to associate stably with other viral proteins, it is most likely that each species exists as a disulphide-linked homodimer. Pulse-chase experiments indicated that gGVL is rapidly assembled as a homodimer prior to both carbohydrate side-chain maturation in the Golgi and proteolytic cleavage. Proteolytic cleavage of full-length gG occurs during or immediately after passage through the Golgi. Secreted and virion-associated species of gG were identified in the closely related virus EHV-1 and were of similar molecular masses to the corresponding EHV-4 gG species.

Glycosylation of a synthetic peptide representing a T-cell determinant of influenza virus hemagglutinin results in loss of recognition by CD4+ T-cell clones.

Synthetic glycopeptides were used to study possible mechanisms for the reduction observed in the response of influenza virus-specific CD4+ T-cells to strains of virus in which amino acid substitution in the hemagglutinin has led to attachment of a carbohydrate side chain. The peptide NCTLIDALLGDPH stimulates vigorous proliferation of hemagglutinin-specific T-cell clones F1-36 and F1-40 but addition of a heptasaccharide, which approaches the size of natural carbohydrate antennae, eliminated the stimulatory capacity of the peptide. This occurs even though the site of carbohydrate attachment at the N-terminal asparagine lies outside the T-cell determinants encompassed by this sequence. A glycopeptide with only two sugar units was stimulatory for F1-36 but not F1-40, suggesting that peptides with a carbohydrate side chain are able to bind to MHC molecules but that approach of the T-cell receptor of certain clones to the glycopeptide-MHC complex is hindered. Loss of T-cell recognition following attachment of a long carbohydrate side-chain to T-cell determinants is not a general finding because attachment of six carbohydrate units to the peptide, NKYVKQNTLKLA, had little or no effect on the stimulation of a T-cell clone specific for this sequence.