Passive Immunization Experiments Research Articles

Passive Immunization with a Polyclonal Antiserum to the Hemoglobin Receptor of Haemophilus ducreyi Confers Protection against a Homologous Challenge in the Experimental Swine Model of Chancroid

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

Universal Vaccine Based on Ectodomain of Matrix Protein 2 of Influenza A: Fc Receptors and Alveolar Macrophages Mediate Protection

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

B-cell epitope prediction for peptide-based vaccine design: towards a paradigm of biological outcomes for global health

Global health must address a rapidly evolving burden of disease, hence the urgent need for versatile generic technologies exemplified by peptide-based vaccines. B-cell epitope prediction is crucial for designing such vaccines; yet this approach has thus far been largely unsuccessful, prompting further inquiry into the underly-ing reasons for its apparent inadequacy. Two major obstacles to the development of B-cell epitope prediction for peptide-based vaccine design are (1) the prevailing binary classification paradigm, which mandates the problematic dichotomization of continuous outcome variables, and (2) failure to explicitly model biological consequences of immunization that are relevant to practical considerations of safety and efficacy. The first obstacle is eliminated by redefining the predictive task as quantitative estimation of empirically observable biological effects of antibody-antigen binding, such that prediction is benchmarked using measures of corre-lation between continuous rather than dichotomous variables; but this alternative approach by itself fails to address the second obstacle even if benchmark data are selected to exclusively reflect functionally relevant cross-reactivity of antipeptide antibodies with protein antigens (as evidenced by antibody-modulated protein biological activity), particularly where only antibody-antigen binding is actually predicted as a surrogate for its biological effects. To overcome the second obstacle, the prerequisite is deliberate effort to predict, a priori, biological outcomes that are of immediate practical significance from the perspective of vaccination. This de-mands a much broader and deeper systems view of immunobiology than has hitherto been invoked for B-cell epitope prediction. Such a view would facilitate comprehension of many crucial yet largely neglected aspects of the vaccine-design problem. Of these, immunodominance among B-cell epitopes is a central unifying theme that subsumes immune phenomena of tolerance, imprinting and refocusing; but it is meaningful for vaccine design only in the light of disease-specific pathophysiology, which for infectious processes is compli-cated by host-pathogen coevolution. To better support peptide-based vaccine design, B-cell epitope predic-tion would entail individualized quantitative estimation of biological outcomes relevant to safety and efficacy. Passive-immunization experiments could serve as an important initial proving ground for B-cell epitope pre-diction en route to vaccine-design applications, by restricting biological complexity to render epitope-prediction problems more computationally tractable.

Attenuated Francisella asiatica iglC mutant induces protective immunity to francisellosis in tilapia

Francisella asiatica is a Gram-negative, facultative intracellular bacteria that causes fish francisellosis. Fish francisellosis is a severe sub-acute to chronic granulomatous disease with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this widespread emergent disease. The goal of this study was to characterize the efficacy of a defined F. asiatica mutant (Δ iglC) as a live attenuated vaccine against subsequent immersion challenge with the wild-type (WT) organism. In previous work, the Δ iglC was found to be attenuated upon intraperitoneal injection and immersion challenges. In vitro, the Δ iglC exhibited reduced growth in tilapia head-kidney derived macrophages, and was significantly attenuated ( p < 0.001) as demonstrated by cytopathogenic and apoptosis assays. In this study, the Δ iglC was tested to determine its ability to protect tilapia against challenge with high doses (lethal dose 80) of WT bacteria. Naïve tilapia vaccinated by immersion with a suspension of the Δ iglC and subsequently challenged with WT F. asiatica were protected (90% mean percent survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the Δ iglC were subsequently found to protect naïve tilapia against high-dose F. asiatica challenge in passive immunization experiments. Significant protection ( p < 0.001) was obtained when fish were passively immunized and challenged with 10 4 and 10 5 CFU/fish of WT F. asiatica; but not when challenged with 10 6 CFU/fish. This is the first report of a defined live attenuated strain providing protection against F. asiatica in fish.

Combination of Pneumococcal Surface Protein A (PspA) with Whole Cell Pertussis Vaccine Increases Protection Against Pneumococcal Challenge in Mice

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wPlow – a new generation vaccine that contains low levels of B. pertussis LPS – conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wPlow vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTPlow protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.

Suppressive and additive effects in protection mediated by combinations of monoclonal antibodies specific for merozoite surface protein 1 of Plasmodium yoelii

BackgroundThe merozoite surface protein (MSP)-1 is a target antigen of protective immunity and a malaria vaccine candidate. The nature of this protective immune response warrants further investigation: although specific antibody is thought to play a major role, the mechanisms of protection are still unclear. Monoclonal antibodies (mAbs) specific for the C-terminus of MSP-1 from Plasmodium yoelii have been shown previously to provide protection against challenge infection when administered by passive immunization to mice. Three protective mAbs were re-examined and, in particular, the effect of combinations of antibodies on the protection provided was studied. It was found that a combination of two antibodies can either provide additive protective effects or result in a suppression of protection. In this report the importance of antibody subclass and epitope specificity in the outcome of these passive immunization experiments are discussed.MethodsThe minimum protective dose (MPD) for each mAb was determined, and then combinations of antibody at their MPD were investigated for their ability to control parasitaemia and promote survival in groups of mice. Mice were inoculated over three days with the MPD and challenged with a blood stage infection of the virulent P. yoelii 17 XL. The resultant parasitaemia was assessed daily on Giemsa-stained blood films. Following the infection the presence of MSP-1 specific antibodies in the sera was monitored, and the proliferative responses of cells in the spleen of protected mice were measured.ResultsCombining antibodies resulted in either an additive effect on protection, with reduced peak parasitaemia and better survival, or resulted in a suppression of protection over that achieved by a single antibody alone. An additive effect was observed when B6 and F5 that have the same isotype and similar fine specificity, were combined. However, a combination of mAb D3, an IgG2a, with either B6 or F5 (both IgG3) suppressed protection, an effect that may have been due to the combination of different isotypes or to the different fine specificity of the antibodies.ConclusionsThese results suggest that a combination of protective antibodies with either the same or different isotypes can produce either an additive or a suppressive effect in passive immunization. This phenomenon may be important in better understanding immunity in this experimental mouse model of malaria.

Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge withStreptococcus pneumoniaein Mice

Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity to Streptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 mug of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcgamma receptor IIB knockout (FcgammaRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcgammaRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcgammaR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcgammaR(s), whereas 5F6 and 7A9 required the inhibitory FcgammaR (FcgammaRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.

Targeting the Extracellular Membrane-Proximal Domain of Membrane-Bound IgE by Passive Immunization Blocks IgE Synthesis In Vivo

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

Immunization with heat‐killed Francisella tularensis LVS elicits protective antibody‐mediated immunity

Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.

Inhibition of replication of primary HIV-1 isolates in huPBL-NOD/Scid mice by antibodies from HIV-1 infected patients

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.

Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-beta-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C).

Antibody-Mediated Immunomodulation: a Strategy To Improve Host Responses against Microbial Antigens

The immunoregulatory properties of antibody have been recognized since the earliest passive immunization experiments, and the potential to modulate an immune response by deliberate immunization with antigen bound by antibody has been demonstrated in numerous instances over the decades ([2][1], [6][2

Doppler-derived mitral deceleration time as a strong prognostic marker of left ventricular remodeling and survival after acute myocardial infarction: Temporelli PL, Giannuzzi P, Nicolosi GL, et al. J Am Coll Cardiol 2004;43:1646–53

In response to infection with the pathogenic ciliate Ichthyophthirius multifiliis, fish produce serum and mucus antibodies that immobilize the parasite in vitro. The antigens responsible for this phenomenon (referred to as immobilization antigens, or i-antigens) are thought to be involved in protective immunity and are being studied in connection with efforts to develop subunit vaccines. Using mammalian antibodies, the i-antigens of Ichthyophthirius have been identified as a family of related surface proteins with Mr's in the 40–60 kDA range. The amino acid sequence deduced from a 1.2 kb cDNA encoding a member of this family predicts a protein with a highly periodic structure characteristic of the i-antigens of the free-living ciliates, Paramecium and Tetrahymena. To date, four distinct immobilization serotypes of I. multifiliis have been identified. Northern hybridization studies indicate that i-antigen genes of Ichthyophthirius are developmentally regulated during the parasite life cycle and are expressed at extraordinarily high levels in the infective stage. Synthesis of i-antigen mRNA transcripts may also be accompanied by novel RNA processing events. A role for the i-antigens in protective immunity is strongly suggested by the results of passive immunization studies with immobilizing monoclonal antibodies (i-mAbs). Following intraperitoneal injection of naïve channel catfish, i-mAbs confer complete protection against an otherwise lethal parasite challenge. In conjunction with ELISA and in vitro immobilization assays, passive immunization experiments indicate that protection requires the presence of antibody at the site of infection (that is, at the surface of fish). The results of these studies are discussed in the light of current knowledge about mechanisms of protection against I. multifiliis, and a model of surface immunity is presented.

Protection against fatal Pseudomonas aeruginosa pneumonia in mice after nasal immunization with a live, attenuated aroA deletion mutant.

Studies of immunity to Pseudomonas aeruginosa have indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection. To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced an aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1, designated PAO1deltaaroA. Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits. Here, we assessed the protective efficacy of immunization with PAO1deltaaroA against acute fatal pneumonia in mice. After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose. Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains. The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup. In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera. However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses. These data suggest that a combination of humoral and cellular immunity is required for protection against P. aeruginosa lung infections, that such immunity can be elicited by using aroA deletion mutants, and that a multivalent P. aeruginosa vaccine composed of aroA deletion mutants of multiple serogroups holds significant promise.

Freshwater fish trypanosomes: definition of two types, host control by antibodies and lack of antigenic variation.

Haemoflagellates of the genus Trypanosoma are prevalent in freshwater fishes and are transmitted by leeches as vectors. As demonstrated by sequence comparisons of nuclear small subunit rRNA genes, trypanosomes isolated from several fish species at different localities can be divided into at least 2 closely related types, designated Type A and Type B. A clone derived from a Type A isolate from carp (Cyprinus carpio) was used to study the anti-parasite immune response in specified pathogen-free outbred carp. Infection leads to an initial rise in parasitaemia in the blood followed by a sharp decline in all fish (acute phase). Thereafter, in some carp, parasites become undetectable both in the blood and in internal organs while, in others, low numbers can be found in the blood for up to 1 year (chronic phase). Fish that have controlled an acute infection with the clone are not only protected against an homologous challenge infection, but also against the infection with parasite lines derived from carp in the chronic phase of infection. Passive immunization experiments with IgM purified from serum of recovered carp indicate that the infection is controlled by antibodies. The anti-parasite antibody level in recovered carp remains high for many months although the parasitaemia is controlled at very low levels and the half life of IgM, t1/2 = 22.5 days, is comparatively short. The effective control of trypanosomes in laboratory infections is in contrast to the high prevalence in natural and farmed freshwater fish populations.

Anti-recombinant V antigen serum promotes uptake of Yersinia enterocolitica serotype 08 by macrophages.

Phagocytosis resistance even in the presence of opsonizing antibodies is a key feature of pathogenic Yersinia spp. Nevertheless, antibodies against the secreted V antigen and the outer membrane protein YadA are known to mediate protection against Y. enterocolitica serotype 08 in a mouse model with intravenous infection. To investigate the impact of anti-V antigen serum on the interaction of Y. enterocolitica and phagocytic cells, gentamicin kill assays and immunofluorescence staining were performed. In contrast to anti-YadA, the presence of V antigen-specific antibodies resulted in an increased uptake of yersiniae by macrophages. The inhibition of phagocytosis by cytochalasin D suppressed the anti-V antigen-mediated uptake. The uptake-promoting effect of anti-V antigen was more distinct for macrophages than for polymorphonuclear leukocytes. The findings of the passive immunization experiments using an orogastric infection model were in agreement with those of cell-culture experiments. In the first 3 days of infection both antisera exhibit no protective effect on the multiplication of the bacteria in the Peyer's patches. Only mice passively immunized with anti-V antigen survived lethal oral infections with Y. enterocolitica serotype 08. Taken together, the results support the assumption that V antigen might be part of the translocation apparatus and that anti-V antigen inhibits the Yop translocation. In addition, antisera against in-frame-deleted recombinant V antigen were generated. Protection experiments using these antisera suggested that the type-specific region (amino acids 225-232) of the V antigen might not be a protective epitope.

Toward an HIV type 1 vaccine that generates potent, broadly cross-reactive neutralizing antibodies.

P ROGRESS IN T HE D EV EL OPM E NT of an effective vaccine for HIV-1 has been gauged in large part by the ability to induce measurable virus-specific CD8 1 cytotoxic T lymphocytes (CTLs) and neutralizing antibodies as lead correlates of a protective immune response. 1±3 Although there has been success in generating low-level CTLs in a subset of vaccinated volunteers in phase I clinical trials, little progress has been made in generating neutralizing antibodies. Part of the problem for neutralizing antibodies is the high degree of genetic variability in the HIV-1 surface gp120 and transm embrane gp41 glycoproteins. 7 Although conserved neutralization epitopes exist, the current repertoire of candidate HIV-1 vaccines fails to generate sufficient antibodies to them. Critical features of the structure of the oligomeric envelope glycoproteins as they exist in their native form on the virus present yet another level of complexity for immunogen design. Most candidate HIV-1 envelope subunit vaccines are based on monomeric gp120 from T cell line-adapted (TCLA) strains of virus. Those vaccines have generated neutralizing antibodies with variable and sometimes potent activity against the vaccine strain, low activity against heterologous TCLA strains, and poor activity against primary isolates. It is conceivable that this narrow specificity, particularly the inability to neutralize primary isolates, will greatly limit the effectiveness of the antibody component of a vaccine. Scientists have been divided on the issue of whether the weak neutralizing antibody response generated by candidate HIV-1 vaccines is a property of the vaccine or the assay. A major argument has been that current assays underestimate neutralization potency, an argument that stems mostly from the fact that primary isolates are generally less sensitive to neutralization than TCLA strains. A concentrated effort has been made to resolve the vaccine-versus-assay issue by focusing mainly on the assay. We now recognize that primary isolates are relatively insensitive to neutralization under a variety of conditions, 27±30 including those that take into account the differential use of CXCR4 and CCR5 as coreceptors. 31±33 These developments, combined with features revealed by the crystal structure of gp120 and gp41, are making it increasingly clear that the dichotomy in neutralization sensitivity between TCLA strains and primary isolates is unrelated to the assay and actually reflects fundamental differences in the structure of the oligomeric envelope glycoproteins of these two categories of virus. Thus, repeated passage of HIV-1 in T cell lines has the consequence of selecting virus variants on which the neutralization epitopes are readily accessible for efficient antibody binding. Some of these epitopes reside in the V3 cysteine±cysteine loop of gp120, which is a major target for neutralizing antibodies on TCLA strains but not primary isolates. The Division of AIDS, Preventive and Vaccine Branch of the National Institutes of Health (Bethesda, MD), convened a workshop in October of 1998 to discuss new strategies to improve the neutralizing antibody responses generated by candidate HIV-1 vaccines. The goals of the workshop were four-fold: (1) consider the advantages and disadvantages of using genetically engineered cell lines to assess a neutralizing antibody response, (2) examine evidence from passive immunization experiments showing that potent neutralization is required for protection, (3) review the structure of gp120 and gp41 revealed by X-ray crystallography as it relates to neutralization and (4) discuss the merits of novel immunization strategies in early phases of development. A summary of this workshop is presented here.

Immunological characterization of a protective antigen of Erysipelothrix rhusiopathiae: identification of the region responsible for protective immunity.

The gene encoding a protective protein antigen of the gram-positive bacterium Erysipelothrix rhusiopathiae, an important veterinary pathogen responsible for erysipelas in swine and a variety of diseases in animals, was cloned and sequenced. The gene encodes a polypeptide of 597 amino acids plus a putative signal sequence of 29 amino acids, resulting in a mature protein with a molecular mass of 69,017 Da. Sequence analysis of the gene product revealed a C-terminal region composed of nine tandem repeats of 20 amino acids and a total sequence that is nearly identical to that of the 64-kDa cell surface protein (SpaA) of the bacterium. Because of this similarity, the protein was designated SpaA.1. In this study, we examined whether the SpaA.1 protein could induce protective antibodies and whether we could identify the region involved in protective immunity. Both the mature SpaA.1 protein and its C-terminal repeat region, but not the N-terminal segment, were expressed in Escherichia coli and purified as a histidine-tagged fusion recombinant protein. Rabbit antiserum raised against the mature SpaA.1 protein passively protected mice from lethal challenge with a virulent homologous strain, Fujisawa-SmR, suggesting that protection is mediated by humoral antibodies. To determine which domain of the SpaA.1 protein is responsible for the observed protection, mice were actively immunized with either the mature SpaA. 1 protein or the C-terminal repeat region and then challenged with Fujisawa-SmR. The result showed that mice immunized with the mature SpaA.1 protein, but not the C-terminal repeat region, were protected, suggesting that the protection-eliciting epitope(s) is located within the N-terminal two-thirds of the SpaA.1 molecule. This was confirmed by passive immunization experiments in which the protective activity of rabbit antiserum, raised against mature SpaA. 1 protein, was not abolished by absorption with the purified recombinant C-terminal repeat region. In addition, antibodies specific for the C-terminal repeat region were unable to protect mice from lethal challenge. These results show that the N-terminal two-thirds of the SpaA.1 molecule may constitute a good vaccine candidate against erysipelas.

Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection.

Infection by live attenuated retroviruses provides excellent protection from challenge with pathogenic viruses in several animal models, but little is known about which immune effectors are necessary for protection. We examined this using adoptive transfer experiments in the Friend virus mouse model. Transfers of immune spleen cells into naive mice conferred complete protection, and transfers of purified lymphocyte subsets demonstrated that this effect required complex immune responses involving CD4+ and CD8+ T cells and also B cells. In addition, passive immunization experiments demonstrated that antibodies alone reduced virus loads but did not prevent infection. These findings may have implications for retroviral vaccine design in general.

Construction and vaccine potential of acapsular mutants of Erysipelothrix rhusiopathiae: use of excision of Tn916 to inactivate a target gene.

We previously showed that acapsular transposon Tn916 mutants of Erysipelothrix rhusiopathiae are avirulent for mice. In this study, we constructed nonreverting acapsular mutants and examined the vaccine potential of the mutants in mice. A representative acapsular transposon mutant, 33H6, was plated on selective agar containing autoclaved chlortetracycline and quinaldic acid, and two tetracycline-sensitive mutants were obtained. Sequence analysis of chromosomal regions of the mutants in which Tn916 had flanked revealed that Tn916 had spontaneously excised from the region and that the six new nucleotides, which were presumably inserted with Tn916 into 33H6 chromosome, substituted for those present at the insertion site. The mutants were confirmed to be devoid of capsular antigen by Western immunoblotting and were nonvirulent for mice (subcutaneous 50% lethal dose [LD50], >10(9) CFU). The safety and efficacy of acapsular mutants for live vaccines was further studied by using one mutant strain, named YS-1. The YS-1 bacteria were cleared from the skin sites of inoculation, livers, and spleens of the inoculated mice by 7 days after subcutaneous (s.c.) inoculation. Mice immunized s.c. with doses ranging from 2 x 10(4) to 2 x 10(8) CFU of strain YS-1 were completely protected against challenge with 100 LD50 of the homologous, highly virulent strain Fujisawa-SmR 21 days postimmunization, and protective immunity conferred by immunization with 2 x 10(8) CFU of the strain lasted for as long as the 3 months of the observation period. In passive immunization experiments, sera collected from mice immunized with strain YS-1 at days 14 and 21 postimmunization provided protection against challenge with Fujisawa-SmR, whereas sera collected at days 4 and 7 did not. Furthermore, specific spleen cell responses to E. rhusiopathiae antigens were observed in mice immunized with strain YS-1, and cross-protection against the antigenically heterologous bacterium Listeria monocytogenes was observed at 7 days after immunization in the mice, suggesting that cell-mediated immunity had been induced. These results suggest that E. rhusiopathiae YS-1 may be a suitable choice for further studies of vaccine efficacy in swine.