Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

BACKGROUND: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors. RESULT: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target. CONCLUSION: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

Immune Profiling to Determine Early Disease Trajectories Associated With Coronavirus Disease 2019 Mortality Rate: A Substudy from the ACTT-1 Trial.

Coronavirus disease 2019 (COVID-19) outcomes are linked to host immune responses and may be affected by antiviral therapy. We investigated antibody and cytokine responses in ACTT-1 study participants enrolled at our center. We studied serum specimens from 19 hospitalized adults with COVID-19 randomized to treatment with remdesivir or placebo. We assessed severe acute respiratory syndrome coronavirus 2 antibody responses and identified cytokine signatures, using hierarchical clustering. We identified no clear immunologic trends attributable to remdesivir treatment. Seven participants were initially seronegative at study enrollment, and all 4 deaths occurred in this group with more recent symptom onset. We identified 3 dominant cytokine signatures, demonstrating different disease trajectories.

Alanine Substitution Inactivates Cross-Reacting Epitopes in Dengue Virus Recombinant Envelope Proteins.

The expansion of the habitat of mosquitoes belonging to the Aedes genus puts nearly half of the world's population at risk of contracting dengue fever, and a significant fraction will develop its serious hemorrhagic complication, which can be fatal if not diagnosed properly and treated in a timely fashion. Although several diagnostic methods have been approved for dengue diagnostics, their applicability is limited in rural areas of developing countries by sample preparation costs and methodological requirements, as well as cross-reactivity among the different serotypes of the Dengue virus and other flavivirus, such as the Zika virus. For these reasons, it is necessary to generate more specific antigens to improve serological methods that could be cheaper and used in field operations. Here, we describe a strategy for the inactivation of cross-reacting epitopes on the surface of the Dengue virus envelope protein through the synthetic generation of recombinant peptide sequences, where key amino acid residues from Dengue virus serotype 1 (DENV-1) and 2 (DENV-2) are substituted by alanine residues. The proteins thus generated are recognized by 88% of sera from Dengue NS1+ patients and show improved serotype specificity because they do not react with the antibodies present in seroconverted, PCR-serotyped DEN-4 infected patients.

Molecular characterization of the ß-amyloid(4-10) epitope of plaque specific Aß antibodies by affinity-mass spectrometry using alanine site mutation.

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the ß-amyloid(1-42) (Aß) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aß-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aß antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aß(4-10) peptide has been identified as an epitope for the polyclonal anti-Aß(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aß-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aß(4-10) peptides upon affinity binding to a monoclonal anti-Aß(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aß(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aß(1-17) to the monoclonal Aß antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aß-specific antibodies with improved therapeutic efficacy.

Identification of a common Ara h 3 epitope recognized by both the capture and the detection monoclonal antibodies in an ELISA detection kit.

Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography-mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens.

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2.

Fine-tuning of T-cell development by the CD3γ di-leucine-based TCR-sorting motif.

The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulation is abolished in thymocytes from CD3γLLAA mice with a mutated CD3γ diL motif, and that CD3γLLAA mice have reduced numbers of thymocytes compared with aged-matched wild-type mice. We found that early thymocyte development at the ß-selection checkpoint is impaired resulting in reduced numbers of double negative (DN) 4 cells in CD3γLLAA mice. This was not caused by reduced proliferation but most probably by increased down-regulation of the antiapoptotic molecule Bcl-2 causing enhanced apoptosis during the transition from the DN3 to the DN4 stage. In contrast, proliferation of immature CD8 single positive (ISP) thymocytes was increased resulting in normal numbers of ISP in CD3γLLAA mice. Despite the normal numbers of ISP, CD3γLLAA mice had reduced numbers of double positive and SP thymocytes indicating that the CD3γ diL motif also affected later stages of T-cell development. In accordance, we found that positive and negative selection, differentiation toward CD4 and CD8 SP T cells and the development of nonconventional T cells were affected in CD3γLLAA mice. In conclusion, our study identifies an important role of the CD3γ diL motif in T-cell development most probably mediated by its fine-tuning of pre-TCR and TCR expression, down-regulation and signaling.

Characterization of lipoteichoic acid structures from three probiotic Bacillus strains: involvement of D-alanine in their biological activity.

Probiotics represent a potential strategy to influence the host's immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with D-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure-activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove D-alanine. The molecular structure of native and modified LTAs was determined by (1)H NMR and GC-MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their D-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on D-alanine substitutions. D-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.

Multiple sclerosis: the elevated antibody response to Epstein-Barr virus primarily targets, but is not confined to, the glycine-alanine repeat of Epstein-Barr nuclear antigen-1.

Patients with multiple sclerosis (MS) have elevated antibodies against Epstein-Barr virus (EBV), but data on the epitope-resolved specificity of these antibodies are scarce. Using a peptide microarray containing 1465 peptides representing 8 full-length EBV proteins, we identified higher (p<0.001) antibody reactivities to 39 EBV-peptides in MS patients (n=29) compared to healthy controls (n=22). Seventeen of the 39 peptides were from EBNA-1 and 13 located within the glycine-alanine repeat of EBNA-1. Further reactivities were directed against EBNA-3, EBNA-4, EBNA-6, VP26, and LMP1. Thus, antibodies against EBV in MS patients primarily target, but are not confined to, the glycine-alanine repeat of EBNA-1.

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production.

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)(+) follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg ), CD19(+) CD1d(high) CD5(high) , CD19(+) CD25(high) forkhead box protein 3 (FoxP3)(+) regulatory B (Breg ) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS(+) GL-7(+) germinal centre B cells and B220(-) CD138(+) plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4(+) T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA.

Engineering improved T cell receptors using an alanine-scan guided T cell display selection system.

T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3ß region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments.

Immunogenicity and cross protective ability of the central VP2 amino acids of infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar L.).

Infectious pancreatic necrosis virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in juvenile salmonids and postsmolt stages of Atlantic salmon (Salmo salar L.) after transfer to seawater. IPN vaccines have been available for a long time but their efficacy has been variable. The reason for the varying immune response to these vaccines has not well defined and studies on the importance of using vaccine trains homologous to the virulent field strain has not been conclusive. In this study we prepared one vaccine identical to the virulent Norwegian Sp strain NVI-015 (NCBI: 379740) (T(217)A(221)T(247) of VP2) and three other vaccine strains developed using the same genomic backbone altered by reverse genetics at three residues yielding variants, T(217)T(221)T(247), P(217)A(221)A(247), P(217)T(221)A(247). These 4 strains, differing in these three positions only, were used as inactivated, oil-adjuvanted vaccines while two strains, T(217)A(221)T(247) and P(217)T(221)A(247), were used as live vaccines. The results show that these three residues of the VP2 capsid play a key role for immunogenicity of IPNV vaccines. The virulent strain for inactivated vaccines elicited the highest level of virus neutralization (VN) titers and ELISA antibodies. Interestingly, differences in immunogenicity were not reflected in differences in post challenge survival percentages (PCSP) for oil-adjuvanted, inactivated vaccines but clearly so for live vaccines (TAT and PTA). Further post challenge viral carrier state correlated inversely with VN titers at challenge for inactivated vaccines and prevalence of pathology in target organs inversely correlated with protection for live vaccines. Overall, our findings show that a few residues localized on the VP2-capsid are important for immunogenicity of IPNV vaccines.

Localization of inhibitory antibodies to the biotin domain of human pyruvate carboxylase.

Pyruvate carboxylase [EC 6.4.1.1] plays an important anaplerotic role in many species by catalyzing the carboxylation of pyruvate to oxaloacetate. To extend our understanding about the structure and function of pyruvate carboxylase (PC), a series of monoclonal antibodies were raised against sheep liver PC and those displaying inhibitory activity were further characterized. The binding epitopes of two monoclonal antibodies that displayed strong inhibitory activity were mapped. Six overlapping fragments of the human enzyme were expressed as thioredoxin fusion proteins in Escherichia coli and subjected to Western blot analysis. Both monoclonal antibodies (MAbs) recognized fragments encompassing the enzyme's C-terminal region, known to contain the structured biotin domain. Through deletion analysis, this domain was determined to be a minimal size of 80 amino acids. Further deletions that disrupted the conformation of the domain abolished antibody binding, indicating these antibodies recognized discontinuous epitopes. To further define the critical residues required for antibody recognition, a model of the domain was produced and an alanine scan performed on selected surface-exposed residues. Our results show that residues encompassing the biotin attachment site, but not biotin itself, are critical for the binding of both antibodies. These data provide a mechanism to explain the inhibitory activity of the antibodies.

Identification of the critical amino acid residues of immunoglobulin E and immunoglobulin G epitopes in ß-lactoglobulin by alanine scanning analysis.

ß-Lactoglobulin represents one of the major allergens causing cow milk allergy. Few studies have clearly evaluated immunological relationships between IgE- and IgG-binding epitopes of ß-lactoglobulin. For characterization of immunological epitopes, peptides of 15 amino acids (AA) in length were synthesized. Immunoglobulin E- and IgG-binding epitopes were immunolabeled with individual sera from cow milk-allergic patients. Alanine scanning of immunodominant epitopes was used to identify the critical AA of IgE- and IgG-binding epitopes. The results showed that 4 IgE-binding epitopes were identified. Our initial data revealed IgE-binding epitopes at AA 17 to 31, AA 72 to 86, AA 92 to 106, and AA 152 to 166. Threonine 20, Met23, and Asp27 are the critical AA of IgE-binding epitopes. Two IgG epitopes were identified, which were located at AA 22 to 36 and AA 127 to 141. The critical AA of IgG-binding epitopes were Leu26 and Val31. Results obtained from this study will provide necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE- or IgG-binding capacity.

Defining TNF-α and IL-1ß induced nascent proteins: combining bio-orthogonal non-canonical amino acid tagging and proteomics.

An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1ß in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio-orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l-azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1ß induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1ß.

Antitumour response of a double mutant of staphylococcal enterotoxin C2 with the decreased affinity for MHC class II molecule.

Staphylococcal enterotoxin C2 (SEC2) is one of the most potent known activators of human T lymphocytes, and recombinant SEC2 shows promising clinical values, but SEC2 can cause food poisoning and toxic shock syndrome in vivo. In this study, site-directed mutagenesis has been used to introduce alanine substitutions at Phe144 and Leu45 in the molecule. The mutant genes were cloned and expressed, and the corresponding proteins were purified by nickel agarose affinity chromatography. We found that the SEC2 mutant proteins could stimulate the proliferation of human peripheral blood lymphocytes and inhibit the growth of tumour cells as native SEC2. Furthermore, flow cytometry assay showed that mSEC2(F44A, L45A) drastically reduced the ability of the toxin to bind to MHC class II. Physiological parameters revealed that mSEC2(F44A, L45A) reduced significantly rat temperature compared with native SEC2 in vivo. Our results clearly suggest that this genetically modified SEC2 protein is less toxic and justifies its further development as a new, safer antitumour superantigen to prevent SEC2 intoxication.

L-Alanine augments rhizobacteria-induced systemic resistance in cucumber.

Bacillus vallismortis strain EXTN-1 is a proven biotic elicitor of systemic resistance in many crops against various pathogens. L: -Alanine (Ala) was tested in cucumber as a chemical elicitor of induced systemic resistance (ISR) against Colletotrichum orbiculare. In the greenhouse, both Ala and EXTN-1 induced significant levels of disease suppression in cucumber against anthracnose. When cucumber plants were treated with EXTN-1 and Ala together, augmentative disease suppression was observed. Experiments with transgenic tobacco plants carrying pathogenesis-related genes fused with the beta-glucuronidase (GUS) reported gene (PR-1a::GUS & PDF 1.2::GUS) showed an enhanced activation of both PR-1a and PDF 1.2 genes upon combined treatment with Ala and EXTN-1. RT-PCR analysis with transgenic (PR-1a or PDF 1.2 over expressing) Arabidopsis plant showed more enhanced expression of resistance genes PR-1a and PDF 1.2 upon combined treatment with Ala and EXTN-1 than either alone. An augmentative ISR effect, when the bacterial elicitor and chemical elicitor were combined together, was confirmed.

Molecular interactions of alanine-rich and proline-rich regions of cell surface protein antigen c in Streptococcus mutans.

INTRODUCTION: Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans, and its cell surface protein antigen c (PAc) is known to be associated with sucrose-independent adhesion to tooth surfaces. PAc is composed of several domains, including an N-terminal signal sequence, an alanine-rich repeat region (A-region), a proline-rich repeat region (P-region), and an anchor region. METHODS: To investigate the functions of each domain, an A-region-deficient mutant strain of S. mutans was constructed, and recombinant PAc and A- and P-region proteins were also constructed. The interactions of each domain with the recombinant proteins were analyzed using surface plasmon resonance spectroscopy with a biomolecular interaction analyzing system. RESULTS: The A-region-deficient mutant strain showed the lowest levels of adherence to saliva-coated hydroxyapatite. Furthermore, findings in an immunoblot assay indicated that the A-region protein reacted strongly with proline-rich proteins in saliva, while the recombinant P-region protein interacted more quickly with PAc than the recombinant A-region protein. CONCLUSION: These results suggest that the A-region has a strong relationship with adhesion to tooth surfaces, while the P-region has a high affinity for PAc.

Immunohistochemical localization of D-alanine to beta-cells in rat pancreas.

A mouse monoclonal antibody against D-alanine (D-Ala) has been raised and the immunohistochemical localization of this D-amino acids in the rat pancreas is visualized. The obtained anti-D-Ala monoclonal antibody has no significant cross-reactivity to all proteinogenic L-amino acids and their D-enantiomers. Using this antibody, immunohistochemical staining was performed on the pancreas, and specific staining for d-Ala has been observed only in the Langerhans islets. To identify the types of D-Ala-immunopositive cells, double staining was carried out with antibodies against D-Ala and pancreatic hormones. Similar immunostaining patterns have been observed for D-Ala and insulin, while D-Ala is hardly co-localized with other hormones (glucagon, somatostatin, and pancreatic polypeptide). These results indicate for the first time that D-Ala is localized to insulin producing beta-cells in mammalian pancreas, suggesting that this D-amino acid would be involved in the regulation of the blood glucose level.

Epitope enhancement of a CD4 HIV epitope toward the development of the next generation HIV vaccine.

Virus-specific CD4+ T cell help and CD8+ cytotoxic T cell responses are critical for maintenance of effective immunity in chronic viral infections. The importance of CD4+ T cells has been documented in HIV infection. To investigate whether a stronger CD4+ T cell response can be induced by modifications to enhance the T1 epitope, the first CD4+ T cell epitope discovered in HIV-1-gp120, we developed a T1-specific CD4+ T cell line from a healthy volunteer immunized with a canarypox vector expressing gp120 and boosted with recombinant gp120. This T1-specific CD4+ T cell line was restricted to DR13, which is common in U.S. Caucasians and African-Americans and very frequent in Africans. Peptides with certain amino acid substitutions in key positions induced enhanced specific CD4+ T cell proliferative responses at lower peptide concentration than the original epitope. This relatively conserved CD4 epitope improved by the epitope enhancement strategy could be a component of a more effective second generation vaccine construct for HIV infection.