Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling.

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

HLA- and genotype-based risk assessment model to identify infantile onset pompe disease patients at high-risk of developing significant anti-drug antibodies (ADA).

In Pompe disease, anti-drug antibodies (ADA) to acid alpha-glucosidase (GAA) enzyme replacement therapy contribute to early mortality. Assessing individual risk for ADA development is notoriously difficult in (CRIM-positive) patients expressing endogenous GAA. The individualized T cell epitope measure (iTEM) scoring method predicts patient-specific risk of developing ADA against therapeutic recombinant human GAA (rhGAA) using individualized HLA-binding predictions and GAA genotype. CRIM-negative patients were six times more likely to develop high ADA titers than CRIM-positive patients in this retrospective study, whereas patients with high GAA-iTEM scores were 50 times more likely to develop high ADA titers than patients with low GAA-iTEM scores. This approach identifies high-risk IOPD patients requiring immune tolerance induction therapy to prevent significant ADA response to rhGAA leading to a poor clinical outcome and can assess ADA risk in patients receiving replacement therapy for other enzyme or blood factor deficiency disorders.

Can we prevent immunogenicity of human protein drugs?

Monoclonal antibodies have proved to be extremely valuable additions to conventional treatment for rheumatic diseases. However, despite the general trend towards "humanisation", these drugs remain immunogenic in clinical settings, baffling drug developers. In principle, humanised and fully human monoclonal antibodies are "self" immunoglobulins and should be tolerated. In this overview, the factors that may influence this process, the nature of immunogenicity and methods to analyse and modify potential immunogenicity are discussed. Finally, novel approaches to "re-induce" immunological tolerance to these proteins, including gene therapy and the recognition of unique regulatory epitopes, are outlined.

Clinical validation of the "in silico" prediction of immunogenicity of a human recombinant therapeutic protein.

Antibodies elicited by protein therapeutics can cause serious side effects in humans. We studied immunogenicity of a recombinant fusion protein (FPX) consisting of two identical, biologically active, peptides attached to human Fc fragment. EpiMatrix, an in silico epitope-mapping tool, predicted promiscuous T-cell epitope(s) within the 14-amino-acid carboxy-terminal region of the peptide portion of FPX. On administration of FPX in 76 healthy human subjects, 37% developed antibodies after a single injection. A memory T-cell response against the above carboxy-terminus of the peptide was observed in antibody-positive but not in antibody-negative subjects. Promiscuity of the predicted T-cell epitope(s) was confirmed by representation of all common HLA alleles in antibody-positive subjects. As predicted by EpiMatrix, HLA haplotype DRB1*0701/1501 was associated with the highest T-cell and antibody response. In conclusion, in silico prediction can be successfully used to identify Class II restricted T-cell epitopes within therapeutic proteins and predict immunogenicity thereof in humans.

Confirmation of immunogenic consensus sequence HIV-1 T-cell epitopes in Bamako, Mali and Providence, Rhode Island.

The design of epitope-driven vaccines for HIV has been significantly hampered by concerns about conservation of vaccine epitopes across clades of HIV. In previous work, we have described a computer-driven method for a cross-clade HIV vaccine comprised of overlapping, highly conserved helper T-cell epitopes or "immunogenic consensus sequence epitopes" (ICS epitopes). Here, we evaluated and compared the immunogenicity of 20 ICS HIV epitopes in ELISpot assays performed using peripheral blood monocytes (PBMC) from HIV-infected donors in Providence, Rhode Island, USA and in Bamako, Mali, West Africa. Each core 9-mer HIV sequence contained in a given consensus peptide was conserved in at least 105 to as many as 2,250 individual HIV-1 strains. Nineteen of the 20 ICS epitopes (95%) were confirmed in ELISpot assays using PBMC obtained from 13 healthy, HIV-1 infected subjects in Providence, and thirteen of the epitopes (65%) were confirmed in ELISpot assays using PBMC derived from 42 discarded blood units obtained at the Central Blood Bank in Bamako. Twelve of the epitopes were confirmed in ELISpot assays performed both in Providence and Bamako. These data confirm the utility of bioinformatics tools to select and design novel vaccines containing "immunogenic consensus sequence" T-cell epitopes for a globally relevant vaccine against HIV; a similar approach may also be useful for any pathogen that exhibits high variability (influenza, HCV, or variola for example). An HIV vaccine containing these immunogenic consensus sequences is currently under development.

De-immunization of therapeutic proteins by T-cell epitope modification.

Many therapeutic proteins in clinical use have been shown to elicit antibody responses which in some cases have been linked to adverse events. Conventional animal models, although convenient, have rarely been predictive of immunogenicity in humans. New methods for predicting the potential immunogenicity of therapeutic proteins are needed. This treatise proposes a new approach which pairs in silico T-cell epitope analysis with in vitro studies. T-cell epitope mapping algorithms such as EpiMatrix can be used to evaluate a candidate therapeutic protein for T-helper epitopes, followed by confirmation of the T-helper epitopes using in vitro methods such as MHC binding assays and T-cell assays. Once these are identified, substitution of key amino acids in the T-cell epitopes may attenuate the immunogenicity of the protein, since modification of the amino acids in anchor position(s) can abrogate binding to human class II MHC molecules and presentation of the peptides, in the context of MHC, to T-helper cells. Following substitution of the key amino acids, immunogenicity of the modified protein can be evaluated in vitro. In parallel, the potential effect of the modifications on the structure of the protein can be evaluated using in silico modeling methods. This multi-step process has been termed DeFT for de-immunization of functional therapeutics. In this article we review the rationale for the approach, provide several retrospective examples that prove the approach in principle, and describe potential applications to therapeutic protein design. The demand for pre-clinical means of evaluating therapeutic proteins is expected to increase with the number of therapeutic proteins and monoclonal antibodies entering the pre-clinical pipeline. Examples provided offer some preliminary proof that the de-immunization approach may improve clinical outcomes.

Analyzing Mycobacterium tuberculosis proteomes for candidate vaccine epitopes.

Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-gamma) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-gamma response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-gamma ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-gamma secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.

Modelling the immunogenicity of therapeutic proteins using T cell epitope mapping.

A new approach to designing therapeutic proteins is emerging, due to an improved understanding of T cell modulation of the immune response and new methods for modelling T cell epitopes using bioinformatics. In silico T cell epitope-mapping using the bioinformatics, when combined with other ex silico means of evaluating MHC-peptide and T cell interaction such as tetramers and HLA transgenic mice, enables the evaluation of dysfunctional immune responses to therapeutic proteins. This approach may even permit researchers to develop means of modulating anticipated adverse effects. The pocket profile method for developing T cell epitope prediction tools is reviewed here, and a comparison between the pocket profile method and the extended anchor method for epitopes restricted by the class II allele HLA DR B*0101 is described.

Rapid determination of HLA B*07 ligands from the West Nile virus NY99 genome.

Defined T cell epitopes for West Nile (WN) virus may be useful for developing subunit vaccines against WN virus infection and diagnostic reagents to detect WN virus-specific immune response. We applied a bioinformatics (EpiMatrix) approach to search the WN virus NY99 genome for HLA B*07 restricted cytotoxic T cell (CTL) epitopes. Ninety-five of 3,433 WN virus peptides scored above a predetermined cutoff, suggesting that these would be likely to bind to HLA B*07 and would also be likely candidate CTL epitopes. Compared with other methods for genome mapping, derivation of these ligands was rapid and inexpensive. Major histocompatibility complex ligands identified by this method may be used to screen T cells from WN virus-exposed persons for cell-mediated response to WN virus or to develop diagnostic reagents for immunopathogenesis studies and epidemiologic surveillance.

From genome to vaccine: in silico predictions, ex vivo verification.

Bioinformatics tools enable researchers to move rapidly from genome sequence to vaccine design. EpiMer and EpiMatrix are computer-driven pattern-matching algorithms that identify T cell epitopes. Conservatrix, BlastiMer, and Patent-Blast permit the analysis of protein sequences for highly conserved regions, for homology with other known proteins, and for homology with previously patented epitopes, respectively. Two applications of these tools to epitope-driven vaccine design are described in this review. Using Conservatrix and EpiMatrix, we analyzed more than 10000 HIV-1 sequences and identified peptides that were potentially immunostimulatory and highly conserved across HIV-1 clades. MHC binding assays and CTL assays have been carried out: 50 (69%) of the 72 candidate epitopes bound in assays with cell lines expressing the corresponding MHC molecule; 15 of the 24 B7 peptides (63%) stimulated gamma-interferon release in ELISpot assays. These results lend support to the bioinformatics approach to selecting novel, conserved, HIV-1 CTL epitopes. EpiMatrix was also applied to the entire 'proteome' derived from two Mycobacterium tuberculosis (Mtb) genomes. Using EpiMatrix, BlastiMer, and Patent-Blast, we narrowed the list of putative Mtb epitopes to be tested in vitro from 1600000 to 3000, a 99.8% reduction. The pace of vaccine design will accelerate when these and other bioinformatics tools are systematically applied to whole genomes and used in combination with in vitro methods for screening and confirming epitopes.

An HLA-directed molecular and bioinformatics approach identifies new HLA-A11 HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected Thais.

Only limited cytotoxic T lymphocyte (CTL) epitope mapping has been done in nonsubtype B HIV-infected persons. We used molecular immunogenetic tools to determine HIV-specific CTL responses in HIV-1 Env subtype E-infected female sex workers (FSWs) from northern Thailand, where more than 50% of the population is HLA-A11 positive. EpiMatrix, a computer-based T cell epitope prediction algorithm, and a manual editing approach were used to predict 77 possible HLA-A11 CTL epitopes in HIV-1, some of which were conserved between subtypes B and E. MHC binding of these peptides was determined in an HLA-A11 stabilization assay, and binding peptides were tested for CTL recognition in eight HLA-A11-positive FSWs. Subtype E versions of known HLA-A2 subtype B HIV epitopes were also tested in four HLA-A2 positive FSWs. CTL responses were detected in all HLA-A11-positive and in three of four HLA-A2-positive persons. Among the 12 FSWs responses to peptides were found to Pol in 9 (75%), Env in 7 (58%), Nef in 5 (42%), and Gag in 5 (42%), and to conserved epitopes in 8 (67%). To identify HLA-A11 CTL epitopes in the absence of prediction tools, it would have been necessary to test almost 3000 10-mer peptides. EpiMatrix and manual predictions reduced this number to 77, of which 26 were MHC binding and 12 were CTL epitopes. Six of these HLA-A11 CTL epitopes have not been previously reported and are located in RT, gp120, and gp41. This report of CTL responses in subtype E-infected individuals defines epitopes that may be useful in HIV pathogenesis or vaccine studies.

HIV clinical trials in correctional settings: right or retrogression?

The right of incarcerated prison and jail inmates to health care is protected by the 8th and the 14th amendments of the Constitution, respectively. Does the right to health care include access to clinical trials? At the time of this writing, clinical trials have become part of the fabric of HIV/AIDS care, allowing patients to participate in studies of new and often lifesaving treatments. Participation in trials can also be dangerous, as illustrated by the recent death of a subject in a gene therapy trial. This danger is compounded by ethical dilemmas that can arise from the large amount of financial support for clinical trials (greater than 75%) that is derived from for-profit corporations. Indeed, clinical trials are the subject of grave concern on the part of the United States Government, which has recently taken steps to shore up human subject safeguards. Following a conference on the conduct of clinical trials in correctional settings, the Office for Human Research Protections suspended prison research conducted by 4 prestigious academic institutions.

HIV infection among incarcerated women: epidemic behind bars.

Approximately 1 in 109 adult women was under the care, custody, or control of adult criminal justice authorities on any given day in 1998, the year for which the most recent statistics on women in correctional institutions are available. Of the 84,400 women who were in prison in 1998, a large percentage--37% in state facilities and 72% in federal prisons--were charged with drug-related offenses. Besides drug use, an additional determinant of HIV infection among incarcerated women may be prior exposure to physical and sexual abuse. Linkages among histories of childhood sexual abuse, physical abuse, drug use, and sex work are believed to explain the disproportionately high prevalence of HIV infection among incarcerated women. Historically, HIV services have had to compete with other demands on correctional budgets for funding and personnel time, even though the correctional health care unit is a unique and highly cost-effective access point for providing HIV prevention and care for high-risk populations of women. Coalition building between correctional staff and medical staff (and, in some cases, departments of public health) has enabled some correctional institutions for women to establish outstanding programs for HIV-infected women. By diagnosing HIV and instituting a plan for treatment, correctional facilities for women can play a critically important role in the reduction of morbidity and mortality among HIV-infected women in high-risk populations.

Prediction of well-conserved HIV-1 ligands using a matrix-based algorithm, EpiMatrix.

This preliminary study was undertaken to identify new human leucocyte antigens (HLA) ligands from human immunodeficiency virus type 1 (HIV-1) which are highly conserved across HIV-1 clades and which may serve to induce cross-reactive cytotoxic T lymphocytes (CTLs). EpiMatrix was used to predict putative ligands from HIV-1 for HLA-A2 and HLA-B27. Twenty-six peptides that were both likely to bind and also highly conserved across HIV-1 strains in the Los Alamos HIV sequence database were selected for binding assays using the T2 stabilization assay. Two peptides that were also highly likely to bind (for A2 and B27, as determined by EpiMatrix) and well conserved across HIV-1 strains, and had previously been described to bind in the published literature, were also selected to serve as positive controls for the assays. Ten new major histocompatibility complex (MHC) ligands were identified among the 26 study peptides. The control peptides bound, as expected. These data confirm that EpiMatrix can be used to screen HIV-1 protein sequences for highly conserved regions that are likely to bind to MHC and may prove to be highly conserved HIV-1 CTL epitopes.

Molecular epidemiology of HIV-1 infection in the Philippines, 1985 to 1997: transmission of subtypes B and E and potential emergence of subtypes C and F.

The molecular epidemiology of HIV-1 infection in the Philippines from 1985 to 1997 was investigated following subtyping of 54 (33 women, 21 men) prospectively collected clinical specimens using the heteroduplex mobility assay (HMA). In contrast with other Asian countries, subtype B accounted for most (70%) of the infections in the population studied, among female commercial sex workers (CSWs, 18 of 28), overseas contract workers (OCWs, 7 of 10), and men who have sex with men (MSM, 8 of 10). However, although viral specimens from HIV-seropositive persons diagnosed before 1993 (n = 16) were all of subtype B, diagnoses in more recent years (1993-present, n = 38) indicate the existence of subtypes E (29%), F (8%), and C (5%) in the population. Since its estimated introduction in the early 1990s, subtype E has accounted for 60% of the infections among female CSWs diagnosed after 1992 (n = 15). This genotype distribution shift occurred in parallel with a shift in transmission focus from the U.S. military bases to the the Philippine national capital region. So far, both events appear to have had no significant effect on the stability of HIV-1 transmission in the country. The recent identification of non-B subtypes in the Philippines may present novel insights on the dynamics of HIV-1 transmission in a high-risk but low-HIV prevalence setting in Asia.