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1.  Identification of an immunodominant neutralizing and protective epitope from measles virus fusion protein by using human sera from acute infection. 
Journal of Virology  1997;71(10):7240-7245.
Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles.
PMCID: PMC192064  PMID: 9311797
2.  Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus. 
Journal of Virology  1995;69(3):1420-1428.
Synthetic peptides representing T- and B-cell epitopes from the fusion (F) protein of measles virus (MV) were tested for their ability to induce a protective immune response against intracerebral challenge with neuroadapted strains of MV and canine distemper virus (CDV) in mice. Of the panel of peptides tested, only a chimeric peptide consisting of two copies of a promiscuous T-cell epitope (representing residues 288 to 302 of MV F protein) synthesized at the amino terminus of a B-cell epitope (representing residues 404 to 414 of MV F protein) was able to induce a protective response against challenge with MV and CDV in inbred mice. The protective response induced by this peptide (TTB) was associated with a significant reduction in mortality, histological absence of acute encephalitis, and greatly reduced titers of virus in the brains of TTB-immune mice following challenge compared with the results for nonimmunized controls. A chimeric peptide comprising one copy of the T-cell epitope and one copy of the B-cell epitope (TB) did not induce a protective response. A comparison of the antibody responses induced by the two chimeras suggested that differences in protective efficacy following immunization may be a result of the higher affinity of the antibody induced by the TTB peptide than that of the antibody induced by the TB peptide. In addition, differences in the immunoglobulin G subclass of the antipeptide antibody responses were observed, and these may play a role in the differences in protection observed. These results indicate that appropriately designed synthetic peptides have potential as vaccines for the induction of cross-reactive protection against morbilliviruses.
PMCID: PMC188728  PMID: 7531779
3.  Benchmarking B-Cell Epitope Prediction with Quantitative Dose-Response Data on Antipeptide Antibodies: Towards Novel Pharmaceutical Product Development 
BioMed Research International  2014;2014:867905.
B-cell epitope prediction can enable novel pharmaceutical product development. However, a mechanistically framed consensus has yet to emerge on benchmarking such prediction, thus presenting an opportunity to establish standards of practice that circumvent epistemic inconsistencies of casting the epitope prediction task as a binary-classification problem. As an alternative to conventional dichotomous qualitative benchmark data, quantitative dose-response data on antibody-mediated biological effects are more meaningful from an information-theoretic perspective in the sense that such effects may be expressed as probabilities (e.g., of functional inhibition by antibody) for which the Shannon information entropy (SIE) can be evaluated as a measure of informativeness. Accordingly, half-maximal biological effects (e.g., at median inhibitory concentrations of antibody) correspond to maximally informative data while undetectable and maximal biological effects correspond to minimally informative data. This applies to benchmarking B-cell epitope prediction for the design of peptide-based immunogens that elicit antipeptide antibodies with functionally relevant cross-reactivity. Presently, the Immune Epitope Database (IEDB) contains relatively few quantitative dose-response data on such cross-reactivity. Only a small fraction of these IEDB data is maximally informative, and many more of them are minimally informative (i.e., with zero SIE). Nevertheless, the numerous qualitative data in IEDB suggest how to overcome the paucity of informative benchmark data.
doi:10.1155/2014/867905
PMCID: PMC4037609  PMID: 24949474
4.  Adherence-Inhibitory Intestinal Immunoglobulin A Antibody Response in Baboons Elicited by Use of a Synthetic Intranasal Lectin-Based Amebiasis Subunit Vaccine▿  
Infection and Immunity  2007;75(8):3812-3822.
We designed an amebiasis subunit vaccine that is constructed by using four peptide epitopes of the galactose-inhibitable lectin heavy subunit that were recognized by intestinal secretory immunoglobulin A (IgA) antibodies from immune human subjects. These epitopes are contained in the region encompassing amino acids 758 to 1134 of the lectin heavy subunit, designated LC3. Baboons (Papio anubis) are natural hosts for Entamoeba histolytica; naturally infected baboons raised in captivity possess serum IgA antibodies to the same four LC3 epitopes as humans. Uninfected, seronegative baboons received four intranasal immunizations at 7-day intervals with the synthetic peptide vaccine (400, 800, or 1,600 μg per nostril) with cholera toxin (20 μg) as the adjuvant. As determined by an enzyme-linked immunosorbent assay (ELISA), each dose of the peptide vaccine elicited antipeptide serum IgA and IgG and intestinal IgA antibody responses in all six immunized baboons by day 28, 7 days after the last immunization (P, <0.01 for each dose compared to the cholera toxin control). The peptide vaccine elicited serum IgG and intestinal IgA antibodies that recognized purified recombinant LC3 protein (P, <0.008 and 0.02, respectively) and native lectin protein (P < 0.01). In addition, an indirect immunofluorescence assay with whole trophozoites (P < 0.01) and Western blot analysis confirmed that serum IgG antibodies from vaccinated baboons recognized native lectin protein on the surfaces of axenic E. histolytica trophozoites or from solubilized amebae. All four synthetic peptides were immunogenic; the vaccine elicited dose- and time-dependent responses, as determined by ELISA optical density readings indicating the production of serum and intestinal antibodies (P, <0.02 for antipeptide and antilectin antibodies). As a positive control, intranasal immunization with purified recombinant LC3 protein with cholera toxin as the adjuvant elicited a serum anti-LC3 IgA and IgG antibody response (P, 0.05 and <0.0001, respectively); however, no intestinal anti-LC3 IgA antibody response was observed (P = 0.4). Of interest, serum IgA and IgG antibodies elicited by the recombinant LC3 vaccine did not recognize any of the four putatively protective LC3 peptide epitopes. Both serum and fecal antibodies elicited by the peptide vaccine exhibited neutralizing activity, as determined by their dose-dependent inhibition of the galactose-specific adherence of E. histolytica trophozoites to Chinese hamster ovary cells in vitro (P, <0.001 for each group of antibodies compared to the control). In summary, a lectin-based intranasal polylysine-linked synthetic peptide vaccine was effective in eliciting an adherence-inhibitory, intestinal antilectin IgA antibody response in baboons. Future studies with the baboon model will determine vaccine efficacy against asymptomatic E. histolytica intestinal infection.
doi:10.1128/IAI.00341-07
PMCID: PMC1952019  PMID: 17526742
5.  AID Activity in B Cells Strongly Correlates with Polyclonal Antibody Affinity Maturation in-vivo Following Pandemic 2009-H1N1 Vaccination in Humans 
PLoS Pathogens  2012;8(9):e1002920.
The role of Activation-Induced Cytidine Deaminase (AID) in somatic hypermutation and polyclonal antibody affinity maturation has not been shown for polyclonal responses in humans. We investigated whether AID induction in human B cells following H1N1pdm09 vaccination correlated with in-vivo antibody affinity maturation against hemagglutinin domains in plasma of young and elderly individuals. AID was measured by qPCR in B cells from individuals of different ages immunized with the H1N1pdm09 influenza vaccine. Polyclonal antibody affinity in human plasma for the HA1 and HA2 domains of the H1N1pdm09 hemagglutinin was measured by antibody-antigen complex dissociation rates using real time kinetics in Surface Plasmon Resonance. Results show an age-related decrease in AID induction in B cells following H1N1pdm09 vaccination. Levels of AID mRNA before vaccination and fold-increase of AID mRNA expression after H1N1pdm09 vaccination directly correlated with increase in polyclonal antibody affinity to the HA1 globular domain (but not to the conserved HA2 stalk). In the younger population, significant affinity maturation to the HA1 globular domain was observed, which associated with initial levels of AID and fold-increase in AID after vaccination. In some older individuals (>65 yr), higher affinity to the HA1 domain was observed before vaccination and H1N1pdm09 vaccination resulted in minimal change in antibody affinity, which correlated with low AID induction in this age group. These findings demonstrate for the first time a strong correlation between AID induction and in-vivo antibody affinity maturation in humans. The ability to generate high affinity antibodies could have significant impact on the elucidation of age-specific antibody responses following vaccination and eventual clinical efficacy and disease outcome.
Author Summary
Antibody affinity maturation is a key aspect of an effective immune response to vaccines, likely to have an impact on clinical outcome following exposure to pathogens. Activation-Induced Cytidine Deaminase (AID) in B cells is a key enzyme involved in antibody class switching and somatic hypermutation, required for antibody affinity maturation. This human study demonstrated for the first time that induction of AID following H1N1pdm09 influenza vaccination directly correlated with in-vivo antibody affinity maturation against the hemagglutinin globular domain (HA1), containing most of the protective targets. Importantly, age differences were found. In younger adults, significant affinity maturation to the HA1 globular domain was observed, which associated with higher initial levels of AID and >2-fold-increase in AID after vaccination. With increased age, a drop in AID activity post-vaccination correlated with lower affinity maturation of the polyclonal antibody responses against the pandemic influenza HA1. However, in a subset of elderly (>65 yr), high affinity antibodies against the HA1 were present prior to vaccination but, in the absence of AID, did not undergo further maturation. Therefore, vaccination of divergent human populations, especially older individuals, should take into consideration their individual AID status and the history of exposure and vaccination against the specific pathogen.
doi:10.1371/journal.ppat.1002920
PMCID: PMC3441753  PMID: 23028320
6.  Influence of epitope polarity and adjuvants on the immunogenicity and efficacy of a synthetic peptide vaccine against Semliki Forest virus. 
Journal of Virology  1993;67(10):5843-5848.
The antibody response to a previously defined B-cell epitope of Semliki Forest virus (SFV) was investigated in male BALB/c (H-2d) mice. The B-cell epitope, located at amino acid positions 240 to 255 of the E2 protein, was linked to an H-2d-restricted T-helper cell epitope of SFV located at positions 137 to 151 of the E2 protein. Colinearly synthesized peptides, of either T-B or B-T polarity, mixed with different adjuvants (the nonionic block copolymer L 180.5, a water-oil-water [W/O/W] emulsion of L 180.5, Montanide, and Q VAC) were used for immunization. Generally, after one booster immunization, high serum antibody titers were measured against either peptide. With Q VAC and W/O/W L 180.5 as adjuvants, the titers of SFV-reactive (nonneutralizing) antibodies were consistently much higher after immunization with the T-B peptide than with the B-T peptide, which was reflected in a higher vaccine efficacy. With these two adjuvants, the survival ratio in T-B peptide-immunized mice was 82%, compared with 8% in B-T peptide-immunized mice. Intermediate results were obtained with the adjuvant Montanide. L 180.5 alone was ineffective in this study. All immunoglobulin G (IgG) isotypes were induced with either adjuvant, but Q VAC was clearly the most effective in inducing IgG2a and IgG2b isotypes with the T-B peptide as the antigen. Subsequently, monoclonal antibodies (MAbs) of IgM, IgG1, IgG2a, IgG2b, and IgG3 subclasses were prepared against the B-cell epitope. These nonneutralizing but SFV-reactive MAbs protected 40 to 80% of mice against a lethal challenge with SFV. Control mice all died. The availability of those antipeptide MAbs allowed competition binding assays with a previously characterized panel of E2-specific MAbs. Binding of enzyme-labeled antipeptide MAbs was very effectively inhibited by two strongly SFV-neutralizing mutually competitive MAbs, suggesting that the linear B-cell epitope (amino acids 240 to 255) is associated with a major neutralization site of SFV.
PMCID: PMC238002  PMID: 7690411
7.  Benchmarking B-Cell Epitope Prediction for the Design of Peptide-Based Vaccines: Problems and Prospects 
To better support the design of peptide-based vaccines, refinement of methods to predict B-cell epitopes necessitates meaningful benchmarking against empirical data on the cross-reactivity of polyclonal antipeptide antibodies with proteins, such that the positive data reflect functionally relevant cross-reactivity (which is consistent with antibody-mediated change in protein function) and the negative data reflect genuine absence of cross-reactivity (rather than apparent absence of cross-reactivity due to artifactual masking of B-cell epitopes in immunoassays). These data are heterogeneous in view of multiple factors that complicate B-cell epitope prediction, notably physicochemical factors that define key structural differences between immunizing peptides and their cognate proteins (e.g., unmatched electrical charges along the peptide-protein sequence alignments). If the data are partitioned with respect to these factors, iterative parallel benchmarking against the resulting subsets of data provides a basis for systematically identifying and addressing the limitations of methods for B-cell epitope prediction as applied to vaccine design.
doi:10.1155/2010/910524
PMCID: PMC2847767  PMID: 20368996
8.  Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus. 
Journal of Virology  1988;62(9):3474-3483.
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.
Images
PMCID: PMC253472  PMID: 2457115
9.  Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues 
Infection and Immunity  1998;66(6):2895-2904.
The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum.
PMCID: PMC108287  PMID: 9596765
10.  Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells 
The FEBS journal  2007;274(7):1747-1758.
Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrPSc). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrPC) has been reported to abolish PrPSc infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrPC, active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP–virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrPC in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrPSc in prion-infected cells. If also effective in vivo, PrP–virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable pri-on-mediated diseases.
doi:10.1111/j.1742-4658.2007.05721.x
PMCID: PMC3792370  PMID: 17313482
immunotherapy; papillomavirus-like particles; prion
11.  Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines. 
Infection and Immunity  1995;63(3):961-968.
Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.
PMCID: PMC173096  PMID: 7532630
12.  A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus 
PLoS Pathogens  2011;7(6):e1002111.
Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.
Author Summary
Neutralizing antibodies are a critical aspect of protection from flavivirus infection. The primary targets of neutralizing antibodies are the envelope (E) proteins incorporated into virions. The neutralizing activity of antibodies is determined by the affinity with which they interact with the virion, and the total number of sites available for binding. In this study, we investigate the impact of dynamic motion of the viral E proteins on antibody-mediated neutralization. Using panels of monoclonal antibodies and immune sera, we demonstrate that the dynamic motion of virions significantly impacts antibody-mediated neutralization of West Nile and dengue viruses by modulating epitope accessibility. Increasing the length of the interactions between antibody and virus resulted in increased neutralization reflecting engagement of epitopes that are not exposed on the surface of the virion in its average state, but instead become accessible through the dynamic motion of E proteins. While examples of the impact of structural dynamics on antibody binding have been described previously, our data suggests this phenomenon plays a role in neutralization by all antibodies that bind the array of E proteins on the virion. Our data identifies epitope accessibility as a critical, yet dynamic, factor that governs the neutralizing activity of anti-flavivirus antibodies.
doi:10.1371/journal.ppat.1002111
PMCID: PMC3128118  PMID: 21738473
13.  Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies. 
Infection and Immunity  1996;64(9):3555-3564.
As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.
PMCID: PMC174263  PMID: 8751899
14.  Insensitivity of Paediatric HIV-1 Subtype C Viruses to Broadly Neutralising Monoclonal Antibodies Raised against Subtype B 
PLoS Medicine  2006;3(7):e255.
Background
A Phase I clinical trial has been proposed that uses neutralising monoclonal antibodies (MAbs) as passive immunoprophylaxis to prevent mother-to-child transmission of HIV-1 in South Africa. To assess the suitability of such an approach, we determined the sensitivity of paediatric HIV-1 subtype C viruses to the broadly neutralising MAbs IgG1b12, 2G12, 2F5, and 4E10.
Methods and Findings
The gp160 envelope genes from seven children with HIV-1 subtype C infection were cloned and used to construct Env-pseudotyped viruses that were tested in a single-cycle neutralisation assay. The epitopes defining three of these MAbs were determined from sequence analysis of the envelope genes. None of the seven HIV-1 subtype C pseudovirions was sensitive to 2G12 or 2F5, which correlated with the absence of crucial N-linked glycans that define the 2G12 epitope and substitutions of residues integral to the 2F5 epitope. Four viruses were sensitive to IgG1b12, and all seven viruses were sensitive to 4E10.
Conclusions
Only 4E10 showed significant activity against HIV-1 subtype C isolates, while 2G12 and 2F5 MAbs were ineffective and IgG1b12 was partly effective. It is therefore recommended that 2G12 and 2F5 MAbs not be used for passive immunization experiments in southern Africa and other regions where HIV-1 subtype C viruses predominate.
Editors' Summary
Background.
AIDS is caused by HIV. By killing the cells of the body's immune system, HIV infection makes people vulnerable to many potentially fatal bacterial and viral diseases. HIV is most commonly spread through unprotected sex with an infected partner but it can also pass from mother to child during late pregnancy or birth, or through breast milk. At least one in four infected women will transmit HIV to their babies if left untreated. But if infected women are treated with drugs that fight HIV—so-called antiretrovirals—during late pregnancy and if breastfeeding does not occur, only one to two babies in 100 will become infected with HIV. In addition, elective Caesarian section has been found to be protective against HIV infection. Implementation of this approach has greatly reduced mother-to-child transmission in developed countries, but most HIV-infected women live in developing countries where access to antiretrovirals is limited. In these cases, treatment of pregnant women (during pregnancy and delivery) and their newborn babies with a single dose of one antiretroviral drug, which can halve HIV transmission, is used, even though WHO/UNAIDS recommends simple antenatal, intrapartum, and postnatal antiretroviral regimens to achieve levels of less than 5% transmission in resource poor settings. These strategies will not have an impact on breastmilk transmission, which accounts for half the transmissions in these settings.
Why Was This Study Done?
One way to reduce breastmilk transmission of HIV might be by “passive immunization.” In this, newborn babies would be injected with HIV-specific antibodies—proteins that stick to molecules on the surface of HIV. Because the virus uses these molecules to invade the baby's immune cells, injected antibodies might stop HIV from the mother becoming established in her offspring. Four antibodies have been made in the laboratory—so-called human monoclonal antibodies—that bind to the surface of HIV subtype B, which is found mainly in Europe and North America, and stop HIV from killing human cells. However, most HIV isolated in Africa is subtype C, so in this study researchers have tested whether these antibodies prevent HIV subtype C killing cells grown in the laboratory. It is important, they argue, that antibodies should be shown to work outside the body before testing passive immunization in babies.
What Did the Researchers Do and Find?
The researchers isolated several subtype C viruses from babies born in Johannesburg, South Africa, and made artificial viruses (known as “pseudotyped” viruses) from them. These artificial viruses could then be used in tests to see whether the human monoclonal antibodies could prevent the viruses infecting human cells in a laboratory test, that is, whether the viruses were “sensitive” to the antibodies. All the viruses were insensitive to two of the antibodies (2G12 and 2F5), and the researchers show that this was because the viruses lacked the specific parts of the HIV surface molecules recognized by these antibodies. Four of the viruses were sensitive to an antibody called IgG1b12, and all were sensitive to antibody 4E10, albeit at high concentrations that might be difficult to achieve in people. Finally, the researchers report that the sensitivity of the viruses was not enhanced by using all four antibodies at the same time.
What Do These Findings Mean?
Given these results, the researchers warn against using 2G12 and 2F5 antibodies for passive immunization to prevent mother-to-child transmission, in particular postnatal transmission, in areas where most people are infected with HIV subtype C viruses. Furthermore, because animal studies have indicated that only combinations of at least three monoclonal antibodies with activity against HIV in laboratory tests provide complete protection against HIV infection, the researchers question whether any clinical trials on passive immunization should be started with currently available antibodies. Their doubts about such trials are heightened by observations that 4E10 and 2F5 react against antigens present on human cells, which might make them unsafe for use in people, although so far no adverse effects have been seen in adults treated with these antibodies. However, these experiments used an artificial laboratory-based assay and it's possible that these antibodies might kill HIV subtype C more effectively in people; other components of the immune system might help them deal with the virus. If clinical studies of these antibodies do go ahead, it is essential that the babies in these trials must be carefully monitored to ensure that the antibodies are safe, and they and their mothers should also be given access to optimal antiretroviral prophylaxis according to WHO/UNAIDS guidelines. In a related PLoS Medicine Perspective paper (http://dx.doi.org/10.1371/journal.pmed.0030259), Miroslaw Gorny1 and Susan Zolla-Pazner discuss the study further and stress the critical need to determine if passive immunization with such antibodies could decrease mother-to-child transmission of HIV, and if so what the best antibodies would be.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030255.
•  National Institute of Allergy and Infectious Diseases fact sheets on HIV infection and AIDS
•  US Department of Health and Human Services information on HIV/AIDS, including clinical guidelines and fact sheets on preventing transmission from mother to child
•  US Centers for Disease Control and Prevention information on HIV/AIDS, including pages on the prevention of mother-to-child transmission
•  MedlinePlus encyclopedia entry on HIV/AIDS
•  Preventing mother-to-child transmission of HIV Web page
Assessment of viruses from seven children with HIV-1 subtype C infection showed generally poor sensitivity to four monoclonal antibodies proposed for a trial of passive immunoprophylaxis to prevent mother-to-child transmission.
doi:10.1371/journal.pmed.0030255
PMCID: PMC1502151  PMID: 16834457
15.  Immunogenicity of synthetic peptides of Haemophilus influenzae type b outer membrane protein P1. 
Infection and Immunity  1995;63(10):3751-3758.
To identify the B- and T-cell epitopes of P1 of Haemophilus influenzae type b, 13 peptides covering 90% of the protein were chemically synthesized. Mouse, guinea pig, and rabbit antisera raised against purified native P1 were tested for their reactivities against the peptides in peptide-specific enzyme-linked immunosorbent assays (ELISAs). Six immunodominant linear B-cell epitopes were mapped to residues 103 to 137, 189 to 218, 248 to 283, 307 to 331, 384 to 412, and 400 to 437 of the mature P1 protein. When P1 peptides were screened for their reactivities with three human convalescent-phase serum specimens, peptides corresponding to residues 39 to 64, 226 to 253, and 400 to 437 reacted strongly with the antisera. Four regions (residues 39 to 64, 226 to 253, 339 to 370, and 400 to 437) contained murine T-cell epitopes. Rabbit antipeptide antisera were tested for their reactivities with the immunizing peptides and P1 protein by ELISA and immunoblots. All anti-P1 peptide antisera except those raised against peptide HIBP1-8 (residues 279 to 312) or HIBP1-8-keyhole limpet hemocyanin conjugate were shown to be specific for their respective immunizing peptides by ELISA. In addition, rabbit antisera raised against the synthetic peptides corresponding to residues 1 to 29, 39 to 64, 103 to 137, 189 to 218, 226 to 253, 248 to 283, 307 to 331, and 400 to 437 of the mature P1 protein recognized the P1 protein from both typeable and nontypeable isolates. These results suggest that these peptides contain epitopes highly conserved among typeable and nontypeable strains of H. influenzae. However, none of the antipeptide antisera have bactericidal activity, nor were they protective against H. influenzae type b in the infant rat model of bacteremia.
PMCID: PMC173527  PMID: 7558276
16.  Immunogenicity of polysaccharides conjugated to peptides containing T- and B-cell epitopes. 
Infection and Immunity  1994;62(3):785-792.
To develop a general model of polysaccharide-peptide vaccine, we have investigated the efficiency of linear peptides derived from protein SR, and adhesin of the I/II protein antigen family of oral streptococci, to act as carriers for two T cell-independent polysaccharides: serogroup f polysaccharide from Streptococcus mutans OMZ 175 (poly f) and Saccharomyces cerevisiae mannan. Peptide 3 (YEKEPTPPTRTPDQ) and peptide 6 (TPEDPTDPTDPQDPSS), accessible on the native SR protein as demonstrated by their reactivity in enzyme-linked immunosorbent assays with rat antisera raised against protein SR, correspond to immunodominant regions of SR. Peptide 3 contains at least one B- and one T-cell epitope, as demonstrated by its ability to induce peptide- and SR-specific antibody responses without any carrier and to stimulate the proliferation of rat lymph node cells primed either with free peptide or native SR, whereas peptide 6 contains only B-cell epitope(s). Peptide 3 was then covalently coupled though reductive amination to either poly f or mannan, and peptide 6 was coupled to poly f. Subcutaneous immunizations of rats with poly f-peptide 3 or mannan-peptide 3 conjugates produced a systemic immunoglobulin M (IgM) and IgG antibody response, and the elicited antibodies reacted with free poly f or mannan, peptide 3, protein SR, and S. mutans or S. cerevisiae whole cells. Rats immunized with poly f-peptide 6 did not develop any antipeptide or anti-SR response. Furthermore, a booster immunization of animals with poly f-peptide 3 or mannan-peptide 3 conjugates induced high titers of anti-peptide 3, anti-poly f, and antimannan antibodies, which occurred quickly. The response is anamnestic for the peptide and the polysaccharides and is characterized by an Ig switch from IgM to IgG. The data presented here confirm that the presence of B- and T-cell epitopes is necessary to induce an anamnestic antipeptide response and that a peptide containing relevant B- and T-cell epitopes can act as a good carrier in improving an antipolysaccharide anamnestic immune response.
PMCID: PMC186184  PMID: 7509317
17.  B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis 
Infection and Immunity  1998;66(2):717-723.
ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.
PMCID: PMC107962  PMID: 9453632
18.  HLA-A*0201-restricted CD8+ T-cell epitopes identified in dengue viruses 
Virology Journal  2012;9:259.
Background
All four dengue virus (DV) serotypes (D1V, D2V, D3V and D4V) can cause a series of disorders, ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Previous studies have revealed that DV serotype-specific CD8+ T cells are involved in controlling DV infection. Serotype cross-reactive CD8+ T-cells may contribute to the immunopathogenesis of DHF/DSS. The aim of the study was to identify HLA-A*0201-binding peptides from four DV serotypes. We then examined their immunogenicity in vivo and cross-reactivity within heterologous peptides.
Methods
D1V-derived candidate CD8+ T-cell epitopes were synthesized and evaluated for their affinity to the HLA-A*0201 molecule. Variant peptides representing heterologous D2V, D3V, D4V serotypes were synthesized. The immunogenicity of the high-affinity peptides were evaluated in HLA-A*0201 transgenic mice.
Results
Of the seven D1V-derived candidate epitopes [D1V-NS4a56–64(MLLALIAVL), D1V-C46–54(LVMAFMAFL), D1V-NS4b562–570(LLATSIFKL), D1V-NS2a169–177(AMVLSIVSL), D1V-NS4a140–148(GLLFMILTV), D1V-NS2a144–152(QLWAALLSL) and D1V-NS4b183–191(LLMRTTWAL)], three peptides [D1V-NS4a140–148, D1V-NS2a144–152 and D1V-NS4b183–191] had a high affinity for HLA-A*0201 molecules. Moreover, their variant peptides for D2V, D3V and D4V [D2V-NS4a140–148(AILTVVAAT), D3V-NS4a140-148(GILTLAAIV), D4V-NS4a140-148(TILTIIGLI), D2V-NS2a144–152(QLAVTIMAI), D3V-NS2a144–152(QLWTALVSL), D4V-NS2a143–151(QVGTLALSL), D2V-NS4b182–190(LMMRTTWAL), D3V-NS4b182–190 (LLMRTSWAL) and D4V-NS4b179–187(LLMRTTWAF)] also had a high affinity for HLA-A*0201 molecules. Furthermore, CD8+ T cells directed to these twelve peptides were induced in HLA-A*0201 transgenic mice following immunization with these peptides. Additionally, cross-reactivity within four peptides (D1V-NS4b183–191, D2V-NS4b182–190, D3V-NS4b182–190 and D4V-NS4b179–187) was observed.
Conclusions
Two novel serotype-specific HLA-A*0201-restricted CD8+ T-cell epitopes (NS4a140-148 and NS2a144–152) and one cross-reactive HLA-A*0201-restricted CD8+ T-cell epitopes which is similar to a previously identified epitope were identified in D1V-D4V. Combining prediction algorithms and HLA transgenic mice is an effective strategy to identify HLA-restricted epitopes. Serotype-specific epitopes would be used to determine the protective role of serotype-specific CD8+ T cells, while cross-reactive epitopes may provide assistance in exploring the role of serotype cross-reactive CD8+ T cells in the immunopathogenesis of DHF/DSS.
doi:10.1186/1743-422X-9-259
PMCID: PMC3546861  PMID: 23121866
Dengue virus; CD8+ T-cell epitope; Immunogenicity
19.  Antigenic and immunogenic epitopes shared by human papillomavirus type 16 and bovine, canine, and avian papillomaviruses. 
Journal of Virology  1991;65(12):6862-6871.
All types of papillomaviruses (PV) share common, so-called group-specific epitopes. To identify the major group-specific epitopes, we immunized 26 guinea pigs or rabbits with purified bovine PV type 1 (BPV), canine PV, or avian PV from the common chaffinch. The resulting hyperimmune sera, as well as a commercially available rabbit antiserum to BPV and seven monoclonal antibodies to BPV, were tested in an enzyme-linked immunosorbent assay with a set of 66 overlapping 20-amino-acid peptides representing the complete sequence of the major capsid proteins (L1 and L2) of human PV type 16 (HPV 16). Sera from the same animals before immunization were used as controls. The minimal reactive epitopes within each peptide were further characterized by testing of truncated peptides. The cross-reactive epitopes were clustered in two regions of L1, an internal region (at positions 171 to 235), which contained three epitopes, and the more reactive region at the carboxy terminus (at positions 411 to 475), which contained six epitopes. The most reactive of the HPV 16 broadly cross-reactive epitopes was a carboxy-terminal epitope which had the sequence DTYRF and which reacted with nine of the antisera to BPV, canine PV, or avian PV, with the commercially available rabbit antiserum to BPV, and also with a mouse monoclonal antibody to BPV. Antipeptide antisera to all of the HPV 16 L1 peptides and to the most antigenically reactive of their truncated analogs were made in guinea pigs. Antipeptide antisera reactive with BPV were obtained for three of the cross-reactive epitopes, and one of these antisera allowed highly sensitive detection of group-specific PV antigen by immunoperoxidase staining.
Images
PMCID: PMC250784  PMID: 1719234
20.  PREDIVAC: CD4+ T-cell epitope prediction for vaccine design that covers 95% of HLA class II DR protein diversity 
BMC Bioinformatics  2013;14:52.
Background
CD4+ T-cell epitopes play a crucial role in eliciting vigorous protective immune responses during peptide (epitope)-based vaccination. The prediction of these epitopes focuses on the peptide binding process by MHC class II proteins. The ability to account for MHC class II polymorphism is critical for epitope-based vaccine design tools, as different allelic variants can have different peptide repertoires. In addition, the specificity of CD4+ T-cells is often directed to a very limited set of immunodominant peptides in pathogen proteins. The ability to predict what epitopes are most likely to dominate an immune response remains a challenge.
Results
We developed the computational tool Predivac to predict CD4+ T-cell epitopes. Predivac can make predictions for 95% of all MHC class II protein variants (allotypes), a substantial advance over other available methods. Predivac bases its prediction on the concept of specificity-determining residues. The performance of the method was assessed both for high-affinity HLA class II peptide binding and CD4+ T-cell epitope prediction. In terms of epitope prediction, Predivac outperformed three available pan-specific approaches (delivering the highest specificity). A central finding was the high accuracy delivered by the method in the identification of immunodominant and promiscuous CD4+ T-cell epitopes, which play an essential role in epitope-based vaccine design.
Conclusions
The comprehensive HLA class II allele coverage along with the high specificity in identifying immunodominant CD4+ T-cell epitopes makes Predivac a valuable tool to aid epitope-based vaccine design in the context of a genetically heterogeneous human population.The tool is available at: http://predivac.biosci.uq.edu.au/.
doi:10.1186/1471-2105-14-52
PMCID: PMC3598884  PMID: 23409948
CD4+ T-cell epitope prediction; Epitope-based vaccination; Immunodominance; MHC (HLA) class II proteins; MHC (HLA) class II polymorphism; Pan-specific; Peptide binding prediction; Peptide vaccination, Specificity-determining residues
21.  Protection against Exotoxin A (ETA) and Pseudomonas aeruginosa Infection in Mice with ETA-Specific Antipeptide Antibodies 
Infection and Immunity  1998;66(11):5551-5554.
Pseudomonas aeruginosa is an opportunistic pathogen that causes serious and sometimes fatal infections in the compromised host, especially in patients with major trauma or thermal injuries. Exotoxin A (ETA) is the major and most lethal virulence factor produced by this ubiquitous microorganism. In a recent study (H. S. Elzaim, A. K. Chopra, J. W. Peterson, R. Goodheart, and J. P. Heggers, Infect. Immun. 66:2170–2179, 1998), we identified two major epitopes, one within the translocation domain (amino acid [aa] residues 289 to 333) of ETA and another within the enzymatic domain (aa 610 to 638), by using a panel of antipeptide antibodies. Synthetic peptides representing these two epitopes induced ETA-specific antibodies which were able to abrogate the cytotoxic activity of ETA, as measured by incorporation of [3H]leucine into 3T3 fibroblasts. In the present study, these antibodies were tested for the ability to provide protection against ETA and infection with a toxin-producing strain of P. aeruginosa in a mouse model. Antibodies to either of the synthetic peptides conferred protection against ETA. Also, when used for immunization, both peptides induced active immunity to ETA in mice. Antibodies to the peptide representing a region within the enzymatic domain of ETA, in combination with the antibiotic amikacin, enhanced the survival of mice infected with a toxin-producing strain of P. aeruginosa. Thus, antipeptide antibodies specific for ETA might be paired with antibiotic treatment for passive immunization of patients suffering from P. aeruginosa infection.
PMCID: PMC108698  PMID: 9784572
22.  Carrier Properties of a Protein Derived from Outer Membrane Protein A of Klebsiella pneumoniae 
Infection and Immunity  1999;67(11):5547-5551.
We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1′). After immunization of mice with the rP40-G1′ conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.
PMCID: PMC96924  PMID: 10531198
23.  Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin. 
Infection and Immunity  1991;59(3):750-757.
The B subunit of Shiga toxin and Shiga-like toxin (SLT-1) and its fragments are potentially immunogenic and may generate protective humoral responses against the action of these toxins. We have analyzed the antibody response of rabbits immunized with pure B subunit of SLT-1 or synthetic fragments of the subunit. The immune response to the native B subunit was found to be largely directed at conformational epitopes. More importantly, rabbits immunized with the B subunit were protected from a lethal challenge with SLT-1, indicating that the B subunit represents an excellent vaccine candidate to counter the effects of Shiga toxin and SLT-1 in humans. Polyclonal antibodies against a synthetic peptide corresponding to residues 28 to 40 of the B subunit neutralized the cytotoxicity of SLT-1 towards Vero cells. This region is thus exposed in the native state of the B subunit. The sequence specificity of other antipeptide antisera also provides clues to the state of folding and assembly of the B subunit. Antisera to synthetic peptides representing the N- and C-terminal regions of the SLT-1 B subunit did not cross-react with native B subunit but strongly recognized denatured forms of the protein. Finally, the monoclonal antibody 13C4 was shown to bind to a discontinuous epitope expressed only on the native form of the protein. These immunological reagents can be used to probe the conformational state of the B subunit and the holotoxin as it relates to their functional properties.
Images
PMCID: PMC258323  PMID: 1705242
24.  Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41 
PLoS Medicine  2008;5(1):e9.
Background
The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.
Methods and Findings
Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.
Conclusions
Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.
Julie Overbaugh and colleagues analyze an HIV strain with high susceptibility to antibody neutralization and identify two gp41 envelope mutations that confer this sensitivity by exposing multiple neutralization epitopes.
Editors' Summary
Background.
In 1984 when scientists identified human immunodeficiency virus (HIV)—the cause of acquired immunodeficiency syndrome (AIDS)—many experts believed that a vaccine against HIV infection would soon be developed. Nearly 25 years later, there is still no such vaccine and with about 2.5 million new HIV infections in 2007, an effective vaccine is urgently needed to contain the AIDS epidemic. Vaccines provide protection against infectious diseases by priming the immune system to deal quickly and effectively with viruses and other pathogens. Vaccines do this by exposing the immune system to an immunogen—a fragment or harmless version of the pathogen. The immune system mounts a response against the immunogen and also “learns” from this experience so that if it is ever challenged with a virulent version of the same pathogen, it can quickly contain the threat. Many vaccines work by stimulating an antibody response. Antibodies are proteins made by the immune system that bind to molecules called antigens on the surface of pathogens. Antibodies that inactivate the invader upon binding to it are called “neutralizing” antibodies.
Why Was This Study Done?
Several characteristics of HIV have hampered the development of an effective vaccine. An “envelope” protein consisting of two subunits called gp120 and gp41 covers the outside of HIV. Many regions of this protein change rapidly, so the antibody response stimulated by a vaccine containing the envelope protein of one HIV variant provides little protection against other variants. However, other regions of the protein rarely change, so a vaccine that stimulates the production of antibodies to these “conserved” regions is likely to provide protection against many HIV variants. That is, it will stimulate the production of broadly neutralizing antibodies. Unfortunately, it has been difficult to find HIV vaccines that do this, because these conserved regions are often hidden from the immune system by other parts of the envelope protein. In this study, the researchers investigate the envelope protein of an HIV-1 variant they have isolated that is highly susceptible to inactivation by antibodies specific for these conserved regions. Comparing the envelope protein of this sensitive virus to closely related envelope proteins that are resistant to neutralization could identify features that might, if included in an envelope protein immunogen, produce a vaccine capable of generating broadly neutralizing antibodies.
What Did the Researchers Do and Find?
The researchers isolated a subtype A HIV-1 variant from a newly infected woman in Kenya that was efficiently neutralized by monoclonal antibodies (antibodies made by cells that have been cloned in the laboratory). These antibodies were specific for several different conserved regions of gp41 and gp120. The isolate was also neutralized by antibodies in blood from HIV-1-infected people. The envelope protein of the sensitive variant was the same as that of a resistant variant isolated at the same time from the woman, except for four amino acid changes in conserved regions of gp41 (proteins are made from long strings of amino acids). Using a technique called site-directed mutagenesis, the researchers introduced these amino acid changes into envelope proteins made in the laboratory and determined that just two of these changes were responsible for the neutralization sensitivity of the HIV-1 variant. The introduction of these two changes into the neutralization resistant variant and into the unrelated envelope sequences of another subtype A (common in Africa) HIV-1 variant and a subtype B HIV-1 (common in Europe and the Western Hemisphere) variant increased the sensitivity of all these viruses to antibody neutralization.
What Do These Findings Mean?
These findings show that two amino acid changes in gp41 of a neutralization-sensitive HIV-1 variant are responsible for the sensitivity of this variant to several neutralizing antibodies. The finding that the inclusion of these changes in the envelope protein of neutralization-resistant HIV-1 variants greatly increases their sensitivity to neutralizing antibodies indicates that the normally shielded regions of the protein are somehow made accessible to antibody by these changes. One possibility is that the amino acid changes might modify the overall shape of the envelope protein, thus exposing multiple, normally hidden regions in the HIV-1 envelope protein to antibodies. Importantly, these findings open up the possibility that the inclusion of these modifications in envelope-based immunogens might improve the ability of vaccines to generate broadly neutralizing antibodies against HIV-1.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050009.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including links to resources dealing with HIV vaccine development
Information is available from Avert, an international AIDS charity, on all aspects of HIV and AIDS, including HIV vaccines
The US Centers for Disease Control and prevention provides information on HIV/AIDS including information on its HIV vaccine unit (in English and some information in Spanish)
The AIDS Vaccine Clearinghouse provides clear information about HIV vaccine science, research and product development
The International AIDS Vaccine Initiative also provides straightforward information about the development of HIV vaccines
doi:10.1371/journal.pmed.0050009
PMCID: PMC2174964  PMID: 18177204
25.  Structural and Thermodynamic Approach to Peptide Immunogenicity 
PLoS Computational Biology  2008;4(11):e1000231.
In the conventional paradigm of humoral immunity, B cells recognize their cognate antigen target in its native form. However, it is well known that relatively unstable peptides bearing only partial structural resemblance to the native protein can trigger antibodies recognizing higher-order structures found in the native protein. On the basis of sound thermodynamic principles, this work reveals that stability of immunogenic proteinlike motifs is a critical parameter rationalizing the diverse humoral immune responses induced by different linear peptide epitopes. In this paradigm, peptides with a minimal amount of stability (ΔGX<0 kcal/mol) around a proteinlike motif (X) are capable of inducing antibodies with similar affinity for both peptide and native protein, more weakly stable peptides (ΔGX>0 kcal/mol) trigger antibodies recognizing full protein but not peptide, and unstable peptides (ΔGX>8 kcal/mol) fail to generate antibodies against either peptide or protein. Immunization experiments involving peptides derived from the autoantigen histidyl-tRNA synthetase verify that selected peptides with varying relative stabilities predicted by molecular dynamics simulations induce antibody responses consistent with this theory. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the development/evolution of humoral immune responses.
Author Summary
In the current paradigm of immune system recognition, T cell receptors bind to relatively short peptide sequences complexed with major histocompatibility complex proteins on the surface of antigen presenting cells, while B cell receptors recognize unprocessed protein structures. Yet, ample data exist showing that peptide immunization can trigger B cell responses targeting both the immunizing peptide and peptidelike motifs contained within intact protein—despite the fact that the folding stability of such peptides is often quite low. Using thermodynamic modeling and the technique of molecular dynamics simulations, this work provides a cogent framework for understanding the relative capacity of inherently unstable peptide structures to faithfully trigger B cell antibody production against specific conformational motifs found in native/intact proteins.
doi:10.1371/journal.pcbi.1000231
PMCID: PMC2577884  PMID: 19023401

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