Protective Humoral Immune Responses Research Articles

Epitope scanning using virtual matrix-based algorithms.

Protein sequence and expression databases (transcriptomes) contain the information required to identify epitopes capable of generating protective immune responses in humans. A key event in the initiation of an immune response against disease is the presentation of antigenic peptide epitopes to T cells by human leukocyte antigen (HLA) molecules. Computational filtering tools that allow the prediction of HLA/epitope interaction can be applied to sequence databases to select for candidate epitopes, thus minimising the subsequent amount of laboratory work. Here, the basic principles of epitope prediction and a summary of the available prediction approaches are presented, with a particular emphasis on the use of algorithms based on virtual HLA-II quantitative matrices, capable of predicting promiscuous HLA-II ligands.

Enhanced type I immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate

DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. When compared with conventional vaccines, however, DNA vaccines often induce lower antibody titers. We have now found that formulation of a DNA vaccine encoding hepatitis B surface antigen with calcium- or aluminum phosphate adjuvants can increase antibody titers by 10–100-fold and decrease the immunogenic dose of DNA by 10-fold. Furthermore, boosting an HBs protein-primed response with the adjuvanted DNA vaccine resulted in a dramatic increase in the HBs-specific IgG2a response reflecting a shift towards a TH1 response. The mechanism by which aluminum phosphate exerts its adjuvant effect is not through increased expression of HBsAg in vivo; rather, the adjuvant appears to increase the number and affinity of HBs peptide antigen-specific IFN-γ and IL-2 secreting T cells.

The search for a vaccine against schistosomiasis--a difficult path but an achievable goal.

The search for an effective vaccine against schistosomiasis, a parasitic disease currently affecting over 200 million people, remains a desirable but as yet challenging and elusive goal. Progress in the area has been relatively slow but research demonstrating the ability of humans to acquire natural immunity to schistosome infection, together with the successful use in animals of attenuated vaccines, supplemented with encouraging results obtained with defined antigens, suggests that development of a vaccine is achievable. Noteworthy also are recent immune correlate findings which shed light on the complex, putatively protective immune responses in humans, which have improved the prospects of success. With the first human clinical trial having been completed with a schistosome vaccine candidate, this review examines current progress aimed at achieving the objective of a safe and effective vaccine for widespread use against schistosomiasis. The review emphasises work undertaken in the author's laboratory and those of his chief collaborators in the search for a vaccine against schistosomiasis japonica, a disease of major public health significance in The People's Republic of China and The Philippines. Schistosomiasis vaccines should not be considered as the panacea for schistosomiasis control as, when available, it is generally envisaged that they would be used as one component of an integrated strategy complementing currently available and effective tools such as chemotherapy, improvements to sanitation, piped water supply, effective sewage draining and health education.

Enhancement of pulmonary clearance of Moraxella (Branhamella) catarrhalis following immunization with outer membrane protein CD in a mouse model.

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.

Enhancement of protective humoral (Th2) and cell-mediated (Th1) immune responses against herpes simplex virus-2 through co-delivery of granulocyte-macrophage colony-stimulating factor expression cassettes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) could in theory attract antigen-presenting cells in muscle following intramuscular DNA immunization, resulting in enhanced antigen-specific immune responses. Thus, such adjuvants could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of GM-CSF cDNA as a vaccine adjuvant for herpes simplex virus (HSV)-2 in a mouse challenge model. GM-CSF cDNA co-injection enhanced levels of specific IgG, IgE and IgA against HSV-2 gD protein significantly higher than gD plasmid vaccination alone. Moreover, GM-CSF co-injection induced a dramatic increase in IgG1 levels, as compared to IgG2a levels, suggesting a Th2 bias in the response. T helper cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by GM-CSF cDNA co-injection. When challenged with a lethal dose of HSV-2, GM-CSF co-injection increased survival rates to 90%, an improvement as compared to gD vaccination alone (60-63%). Furthermore, GM-CSF cDNA co-injection reduced herpetic lesions and resulted in a faster recovery from lesions. These data indicate that GM-CSF cDNA enhances both humoral and cellular immune responses and enhances vaccine efficacy, resulting in reduced HSV-2-derived morbidity as well as mortality.

Control of Furunculosis and Enteric Redmouth Disease in Sea-Run Atlantic Salmon Broodstock in the Connecticut and Merrimack Rivers

Abstract Adult sea-run Atlantic salmon Salmo salar captured and transported to Richard Cronin National Salmon Station (Sunderland, Massachusetts), Nashua National Fish Hatchery (Nashua, New Hampshire), and Whittemore State Fish Hatchery (Waterford, Connecticut) during 1986–1992 were treated with oxolinic acid and a bacterin. The bacterin was developed against furunculosis and enteric redmouth disease. Among the 2,552 fish that were treated since 1986, 362 died and 65 (18%) of those fish had furunculosis. Among 206 untreated fish that were maintained as controls, 109 died and 63 (57.8%) had furunculosis. The reduction in mortality could not be attributed to either vaccine or antibiotic alone without further study. A 3-year study was designed to investigate if adult Atlantic salmon, undergoing the stress of migration, handling, and spawning, could mount a protective humoral immune response. Although the salmon were able to produce an agglutinin response, evidence was not found for production of a protective...

Development of novel influenza virus vaccines and vectors.

Approaches to improve the efficacy of the current (killed) influenza virus vaccines include the generation of cold-adapted and genetically engineered influenza viruses containing specific attenuating mutations. It is hoped that these genetically altered viruses, in which the hemagglutinin and neuraminidase genes from circulating strains have been incorporated by reassortment, can be used as safe live influenza virus vaccines to induce a long-lasting protective immune response in humans. In addition, genetically engineered influenza viruses may provide a means for expressing foreign antigens. Immunization of mice with recombinant influenza and vaccinia viruses expressing specific antigens of Plasmodium yoelii resulted in a dramatic protective immune response against malaria in this model. Mice immunized with recombinant influenza viruses expressing human immunodeficiency virus (HIV) epitopes generated long-lasting HIV-specific serum antibodies and secretory IgA in the secretory nasal, vaginal, and intestinal mucosa. These results suggest that genetically engineered influenza viruses may be developed for use as live virus vaccines against influenza as well as other diseases.

Human T cell responses induced by vaccination with Mycobacterium bovis bacillus Calmette-Guérin.

Many aspects of the widely used bacillus Calmette-Guérin (BCG) vaccine against tuberculosis are still the subject of controversy. There is a huge variation in efficacy from one clinical trial to another and no relationship between vaccine-induced skin test conversion and subsequent protection. We have studied in vitro cell-mediated immune responses primed by BCG vaccination in 22 healthy Danish donors with different levels of in vitro purified protein derivative (PPD) reactivity before vaccination. The study demonstrated a markedly different development of reactivity to mycobacterial Ags depending on the prevaccination sensitivity to PPD. Previously sensitized donors mounted a potent and highly accelerated recall response within the first week of BCG vaccination. Nonsensitized donors, in contrast, exhibited a gradually increasing responsiveness to mycobacterial Ags, reaching maximal levels between day 56 and 365 postvaccination. The recognition of different classes of Ags were induced in a stepwise manner: culture filtrate Ags were recognized 1 wk postvaccination followed by cell wall, membrane, and the cytosolic Ag fraction. The T cell response primed by BCG vaccination was characterized as a CD4 response with a Th1-like cytokine pattern and substantial levels of Ag-specific cytotoxicity. The specificity of the T cell response generated was broad and directed to a range of culture filtrate Ag fractions. The study shows that BCG vaccination of previously nonsensitized donors can provide important data on potentially protective immune responses in humans and suggest a careful evaluation of prevaccination sensitivity when investigating vaccine-induced immunity.

Secreted Antigens and Immune Responses to <i>Mycobacterium tuberculosi</i><i>s</i>

Recent information from several laboratories points to proteins secreted from live Mycobacterium tuberculosis as being involved in protective immunity. We have studied protein release from M. tuberculosis during growth and have defined a short-term culture filtrate enriched with secreted antigens and with a minimal content of autolytic products. Several components in this filtrate have been characterized and are presently being studied in animal models of tuberculosis. In the mouse model, protection against tuberculosis is mediated by a long-lived population of memory T cells directed against secreted molecules, and the antigens ESAT-6 and the 85 complex are important key targets for this response. The specificity of the protective immune response in humans is less well defined, but our studies indicate that antigen-specific CD4+ cytotoxic T cells and memory T cells secreting interferon-γ (IFN-γ) are involved. The IFN-γ response is directed against a range of the culture filtrate proteins with a preferential recognition of low molecular mass antigens.

The Role of Somatic Mutation in the Generation of the Protective Humoral Immune Response against Vesicular Stomatitis Virus

During most clinically relevant infections with cytopathic viruses, neutralizing antibodies are generated early, i.e., within the first week of infection. As early as 4 days after immunization of mice with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, hybridomas could be isolated that secreted virus-neutralizing IgGs. Such antibodies were devoid of somatic mutations, showed high binding avidities (∼10 9 M −1), and used V gene fragments predominantly belonging to the V HQ52 and V K19–28 families. In contrast, most secondary and hyperimmune response IgGs isolated 12 and 150 days after infection used several additional V gene combinations. These, which used the V HQ52/V K19–28 combination of early IgGs, were point mutated but showed only marginally enhanced binding avidities. Since all V HQ52/ V K19–28-positive IgGs bound to one subsite within the major antigenic site of VSV-G irrespective of the presence or absence of somatic point mutations, fine specificity diversification of secondary and hyperimmune responses was achieved by newly appearing V gene combinations.

T cell proliferative responses to five human cytomegalovirus proteins in healthy seropositive individuals: implications for vaccine development.

Cell-mediated immunity plays an important role in the host response against human cytomegalovirus (HCMV) infection. For the development of HCMV subunit vaccines it is essential to identify which HCMV proteins can induce protective immune responses in humans. We have studied the T cell proliferative responses to five HCMV proteins (IE1, IE2, pp71, gpUL18 and gB). These five proteins were produced using the maltose-binding protein (MBP) fusion protein system. T cell proliferative responses in 23 seropositive and six seronegative individuals were evaluated. None of the six seronegative individuals showed significant responses to any of the proteins. Of the 23 seropositive individuals, five responded to all five proteins, 14 responded to between one and four proteins and four responded to none of the proteins. The most commonly recognized proteins were gB (17/23, 74%) and IE2 (16/23, 70%). pp71 and IE1 were recognized by 10 of 23 (43%) individuals. Nine of 22 (41%) individuals tested responded to gpUL18, providing evidence that this protein is produced during infection. Our data indicate that a subunit vaccine composed of gB alone may not be sufficient to induce protective immunity in all individuals. The combination of two or three proteins may be more efficient as a potential vaccine.

Hepatitis B Surface Antigen Variation and Protective Immunity

Hepatitis B surface antigen (HBsAg) particles consist predominantly of a glycoprotein of 226 amino acids which bears the B-cell epitopes important for the induction of protective antibody responses in humans. It has been clearly shown that the region between residues 120 and 150 of the S protein represents the a determinants common to all hepatitis B virus (HBV) isolates and is exposed on the surface of the HBV particle. Anti-a antibodies protect adults against the majority of infections irrespective of the subtype of the wild-type virus. Occasional examples of infection positive for anti-HBs antibodies have been associated with the emergence of HBV variants. In particular, asymptomatic infections have been described in vaccinated children, an observation which is associated with an amino acid change in a domain critical for anti-HBs binding. Variation in amino acid sequence is also found within the preS amino terminal extensions of the S protein, although these do not correlate with subtypic variations among the S-antigenic domains. There is no direct evidence that preS determinants per se may stimulate a protective immune response in humans, although the hepatocyte attachment domain is located in the preS1 region which is conserved between HBV isolates. The inclusion of preS specificities augments anti-HBs responses in an experimental animal; however, at the present time it is unclear as to how this may best be exploited in improving hepatitis B vaccines for human use. Variability in HBV envelope proteins has implications for the design of vaccination programmes and the diagnosis of HBV infections; however, the low frequency of HBV variants emerging in the face of increasing levels of herd immunity to hepatitis B at the present time means that the extension of immunization programmes using existing vaccines remains a priority.

Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi.

The bacillus Calmette-Guerin (BCG) is a live attenuated strain of Mycobacterium bovis which offers potential advantages as a vector for mucosal delivery of antigens. Recombinant BCG elicits protective humoral immune responses to a variety of antigens. Furthermore, BCG binds specifically to microfold cells present in the epithelium overlying lymphoid follicles throughout the mucosal immune system. Here we show that a single intranasal vaccination with recombinant BCG expressing the outer-surface protein A antigen from B. burgdorferi results in a prolonged (more than one year) protective systemic IgG response and a highly sustained secretory IgA response which is disseminated throughout the mucosal immune system. Furthermore, intranasal immunization induces marked, organized lymphocyte accumulation in the proximal nasopharyngeal lymphoid tissue as well as at distal mucosal sites; the appearance and persistence of lymphoid aggregates correlates with the secretory immune responses. Thus intranasal immunization with recombinant BCG is a powerful method for inducing long-lasting secretory and systemic immune responses.

Developmentally regulated localization and phosphorylation of SmIrV1, a Schistosoma mansoni antigen with similarity to calnexin.

Potential molecular targets of a protective humoral immune response against schistosomiasis have previously been identified based on their enhanced immunogenicity in mice vaccinated with irradiated cercaria as compared to chronically infected mice. One of these antigens, IrV1, has been molecularly cloned and its sequence shown to be similar to the molecular chaperone calnexin. In this investigation, we partially characterized IrV1 from different developmental stages of the schistosome. Immunoprecipitation studies with antibodies raised against a portion of recombinant IrV1 demonstrated its presence in cercaria, schistosomula, and adult worms with an apparent molecular mass on SDS-polyacrylamide gel electrophoresis of 90 kDa. There was an approximate 6-fold increase in protein expression level during the cercaria to schistosomula transformation. Consistent with a potential role as a molecular chaperone, IrV1 was associated with several metabolically labeled proteins in co-immunoprecipitation studies with the adult worm tegumental fraction. Similar to calnexin, IrV1 was metabolically labeled with 32P in adult worms on serine and threonine residues and was one of the major phosphoproteins of this stage. This phosphorylation was developmentally regulated and coincided with the transformation of cercaria into schistosomula. The localization was also stage-specific as IrV1 was transported from internal regions of cercaria to the outer tegumental layer of schistosomula. The presence of IrV1 on the surface of schistosomula, an unprecedented localization for this family of endoplasmic reticulum proteins, supports additional studies of the immunoprophylactic potential of this molecule.

Immune responses induced by prototype vaccines for AIDS in rhesus monkeys.

A battery of assay systems was used to profile both humoral and cell-mediated immune responses induced by immunization with candidate vaccines consisting of recombinant simian immunodeficiency virus (SIV) glycoproteins rgp110 (nondenatured) with SAF-M adjuvant (gp110 + SAF-M) or rgp140 (denatured) with Freund's adjuvant (gp140 + FA). All of the monkeys became infected after intravenous challenge. However, 16 days following infection, viral antigenemia was reduced in both groups of vaccinates compared to controls. After 23 days antigenemia in the gp110 + SAF-M group remained at the same level as on day 16, whereas antigenemia in the gp140 + FA group was significantly reduced further than the level observed on day 16. Both vaccines induced blastogenic responses in PBMC cultures stimulated with rgp140, which decreased after repeated immunizations. Both vaccines induced high ELISA titers of IgG antibody against rgp140 that were equivalent to the titers in asymptomatic long-term survivors (LTSs). gp110 +/- SAF-M induced high titers of neutralizing antibody. In contrast, gp140 + FA failed to induce neutralizing antibody, suggesting that the natural conformation of the antigen may be essential for the induction of neutralizing antibody. High titers of antibodies capable of complement-mediated cytolysis (ACC) were induced by gp110 + SAF-M, whereas minimal ACC antibodies were induced by gp140 + FA. In spite of high titers of antibodies by ELISA, neither gp110 + SAF-M nor gp140 + FA vaccines induced detectable levels of antibody capable of antibody dependent cell-mediated cytolysis (ADCC). Detectable amounts of MHC class I-restricted, CD8+ cytotoxic T lymphocytes (CTLs) were not induced in immunized monkeys before challenge. After challenge and infection, antibody responses to glycoprotein (detected by ELISA and ACC) as well as glycoprotein-specific CTLs were induced in gp140 + FA vaccinates at levels higher than in nonimmunized control animals, indicating a priming effect by gp140 + FA immunization. No priming effect for ADCC antibody induction was observed in monkeys vaccinated with either gp110 + SAF-M or gp140 + FA. Rhesus monkey groups immunized with two different SIV envelope vaccines differed regarding potentially protective humoral and cell-mediated immune responses. The physical state of the immunogens, the type of adjuvant used, and/or the immunization protocol apparently affected these responses in both a qualitative and quantitative manner.

An antibody- and synthetic peptide-defined rubella virus E1 glycoprotein neutralization domain.

We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets. Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J. S. Wolinsky, M. McCarthy, O. Allen-Cannady, W. T. Moore, R. Jin, S. N. Cao, A. Lovett, and D. Simmons, J. Virol. 65:3986-3994, 1991). Both of these MAbs neutralize RV infectivity. A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain. One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA. Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239). Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV. The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity. These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.

Antibody to Rmp (outer membrane protein 3) increases susceptibility to gonococcal infection.

The severe adverse effects of gonococcal infection on human fertility suggests that Neisseria gonorrhoeae would exert powerful selection for the development of a protective immune response in humans. N. gonorrhoeae is an obligate human pathogen and must persist in humans to survive. Since it is an ecologically successful organism, it must have evolved strategies to evade any human immune response it elicits. In a longitudinal study among 243 women working as prostitutes and experiencing frequent gonococcal infection, younger women, women with HIV infection, and women with antibody to the gonococcal outer membrane protein 3 (Rmp) were at increased risk of infection (adjusted odds ratio 3.4, CI95% 1.1-10.4, P < 0.05). Rmp is highly conserved in N. gonorrhoeae and the blocking of mucosal defences may be one of its functions. As similar proteins occur in many gram negative mucosal pathogens, the enhancing effect of such proteins may be a general strategy whereby bacteria evade human immune responses.

Identification of Conformational Epitopes of the BPV-1 Capsid Recognized by Competitive Inhibition of Sera from Infected or Immunized Animals

Conformational papillomavirus (PV) capsid epitopes are the major targets of neutralizing antibodies. BPV1 virion surface epitopes were analyzed for type-specificity, induction of neutralization, and topographical location with antibodies from selected humans, and BPV1 or -2 immunized or infected cattle, rabbits and mice. Rabbit sera produced against deer PV (DPV) or BPV2 and human sera reacted with intact BPV1 in ELISA, indicating the presence of cross-reactive conformational epitopes on the surface of BPV1 virions. However, competitive binding assays performed on intact BPV1 capsids using unlabeled and biotin-conjugated antibodies revealed that the conformational epitopes of BPV1 recognized by rabbit, bovine and human antibodies were topographically different. Only hyperimmune sera to intact BPV1, BPV2 or DPV neutralized BPV1-induced focus formation of C127 cells with the highest neutralizing titers observed for bovine and rabbit antibodies produced against intact BPV1. Our study showed the complexity of conformational capsid epitopes of BPV1 and their importance for inducing a protective humoral immune response in animals inoculated or immunized with BPV1.

Immunologic and genetic characterization of S180, a cell line of murine origin capable of growing in different inbred strains of mice

Some of the immunologic and genetic properties of the cell line S180 have been examined. These cells grew without restrictions in the peritoneal cavity of different inbred strains of mice and invariably killed the animals. With Northern blots it was demonstrated that S180 cells contained class I mRNAs but failed to transcribe B2m genes. However, under experimental conditions, a protective humoral immune response mediated by cytotoxic antibodies and complement against S180 cells was obtained through non-H-2 antigens in C57BL/6J mice.

Protective Humoral Immune Responses to the Human Immunodeficiency Virus Induced in Immunized Pigs: A Possible Source of Therapeutic Immunoglobulin Preparations

The human immunodeficiency virus (HIV-1) induces progressive and fatal disease in infected hosts. Initially the human immune response appears to control HIV infection. This hypothesis is supported by the long latency period observed during HIV infection prior to development of the active disease state. Similarly the observation of fetal protection from HIV infection in some pregnant women who have high titered neutralizing antibody responses to the virus underscores the importance of the humoral response to HIV in limiting infection. Therefore antibody replacement therapy is likely to provide substantial clinical benefits in this and other infected populations. However, currently there is no safe source for human antibodies with the desired protective qualities necessary for passive immune therapies. For a passive immune therapy to be valid it must protect against a diverse collection of viral isolates. Such a task is likely to require a complex mixture of human antibodies or human substitute antibodies which are available in large quantities and target conserved regions of the viral envelope, such that protection from diverse isolates are realized. Porcine products have been used extensively in many human therapeutic replacement regimens. Their use is primarily due to genetic similarity of the two species at the amino acid level which results in a high acceptance of grafted prosthetics and excellent tolerance of repeatedly administered biologicals. Accordingly we have examined the immunoglobulin responses to the Human Immunodeficiency Virus (HIV-1) of the Yorkshire mixed breed pig. Immunized animals developed significant humoral immunity as judged by ELISA, Western blot, radioimmunoprecipitation, flow microfluorimetry as well as in functional assays including neutralization and syncytia inhibition. The high neutralizing activity obtained and the immunological similarity between human and porcine immunoglobulin suggests that further investigation into the use of porcine immunoglobulin as human replacement antibodies for the therapeutic treatment of HIV is warranted.