Passive immunization with a multicomponent vaccine against conserved domains of apical membrane antigen 1 and 235-kilodalton rhoptry proteins protects mice against Plasmodium yoelii blood-stage challenge infection

During malaria parasite invasion of red blood cells, merozoite proteins bind receptors on the surface of the erythrocyte. Two candidate Plasmodium yoelii adhesion proteins are apical membrane antigen 1 (AMA1) and the 235-kDa rhoptry proteins (P235). Previously, we have demonstrated that passive immunization with monoclonal antibodies (MAbs) 45B1 and 25.77 against AMA1 and P235, respectively, protects against a lethal challenge infection with P. yoelii YM. We show that MAb 45B1 recognizes an epitope located on a conserved surface of PyAMA1, as determined by phage display and analysis of the three-dimensional structure of AMA1, in a region similar to that bound by the P. falciparum AMA1-specific inhibitory antibody 4G2. The epitope recognized by 25.77 could not be assigned. We report here that MAbs 45B1 and 25.77 also protect against challenge with the nonlethal parasite line 17X, in which PyAMA1 has a significantly different amino acid sequence from that in YM. When administered together, the two MAbs acted at least additively in providing protection against challenge with the virulent YM parasite. These results support the concept of developing a multicomponent blood-stage vaccine and the inclusion of polymorphic targets such as AMA1, which these results suggest contain conserved domains recognized by inhibitory antibodies.     

Immunization with recombinant Plasmodium yoelii merozoite surface protein 4/5 protects mice against lethal challenge

Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5), expressed as a recombinant protein, was highly effective at protecting mice against lethal challenge with P. yoelii. There was a significant correlation between prechallenge antibody levels and peak parasitemia, suggesting that the homologues of PyMSP4/5 in Plasmodium falciparum are promising components of a subunit vaccine against malaria.     

Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.     

Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major

The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.     

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.     

Characteristics of the protective response of BALB/c mice immunized with a purified Plasmodium yoelii schizont antigen

The response of BALB/c mice to immunization with a 230,000 mol. wt protective antigen purified from the blood stages of Plasmodium yoelii was analysed. The protective response to immunization was adjuvant-dependent, and saponin was found to be the most effective adjuvant tested. The intraperitoneal route was found to be superior to the subcutaneous route for protective immunization. Maximal protection was achieved using two immunizations with greater than 12.5 micrograms of antigen plus saponin, but even under these conditions challenge infection developed normally for 5 days, followed by the appearance of crisis forms and rapid clearance of parasites. Effective immunization induced high antibody titres, but serum from immunized mice was not protective on passive transfer. On the other hand, hyperimmune serum from mice recovered and rechallenged with live P. yoelii was protective on passive transfer, even though the titre of antibody specific for the 230,000 mol. wt antigen in this serum was the same as that in the serum from mice immunized with the purified antigen. It was concluded that the 230,000 mol. wt antigen may provide protection against P. yoelii via the cell-mediated immune effector pathway described by Playfair et al. (1979), and that hyperimmune serum contains protective antibodies specific for P. yoelii antigens other than the 230,000 mol. wt antigen.     

Genetic immunization of BALB/c mice with a plasmid bearing the gene coding for a hybrid merozoite surface protein 1-hepatitis B virus surface protein fusion protects mice against lethal Plasmodium chabaudi chabaudi PC1 infection

The genetic immunization of rodents with a plasmid coding for a Plasmodium chabaudi merozoite surface protein 1 (C terminus)-hepatitis B virus surface fusion protein (pPcMSP1(19)-HBs) provided protection of mice against subsequent lethal challenge with P. chabaudi chabaudi PC1-infected red blood cells. The percentage of survivor mice was higher in DNA-immunized mice than in animals immunized with a recombinant rPcMSP1(19)- glutathione S-transferase fusion protein administered in Freund adjuvant. In all mice immunized with the pPcMSP1(19)-HBs, a Th1-specific response, including the production of anti-MSP1(19)-specific immunoglobulins predominantly of the immunoglobulin G2a subtype and reacting almost exclusively against discontinuous epitopes, was elicited. The coinjection of Th1-type cytokine-expressing plasmids (gamma interferon, interleukin-2, and granulocyte-macrophage colony-stimulating factor) mostly abolished protection and boosting of MSP1(19)-specific antibodies. The inclusion of a lymph node-targeting signal did not significantly increase protection. These data provide further evidence that MSP1(19)-HBs DNA constructs might be useful as components of a genetic vaccine against the asexual blood stages of Plasmodium.     

Allele specificity of naturally acquired antibody responses against Plasmodium falciparum apical membrane antigen 1

Antibody responses against proteins located on the surface or in the apical organelles of merozoites are presumed to be important components of naturally acquired protective immune responses against the malaria parasite Plasmodium falciparum. However, many merozoite antigens are highly polymorphic, and antibodies induced against one particular allelic form might not be effective in controlling growth of parasites expressing alternative forms. The apical membrane antigen 1 (AMA1) is a polymorphic merozoite protein that is a target of naturally acquired invasion-inhibitory antibodies and is a leading asexual-stage vaccine candidate. We characterized the antibody responses against AMA1 in 262 individuals from Papua New Guinea exposed to malaria by using different allelic forms of the full AMA1 ectodomain and some individual subdomains. The majority of individuals had very high levels of antibodies against AMA1. The prevalence and titer of these antibodies increased with age. Although antibodies against conserved regions of the molecule were predominant in the majority of individuals, most plasma samples also contained antibodies directed against polymorphic regions of the antigen. In a few individuals, predominantly from younger age groups, the majority of antibodies against AMA1 were directed against polymorphic epitopes. The D10 allelic form of AMA1 apparently contains most if not all of the epitopes present in the other allelic forms tested, which might argue for its inclusion in future AMA1-based vaccines to be tested. Some important epitopes in AMA1 involved residues located in domain II or III but depended on more than one domain.     

Immunization of mice against blood-stage Plasmodium yoelii malaria with isoelectrically focused antigens and correlation of immunity with T-cell priming in vivo.

Mice were immunized with lethal Plasmodium yoelii blood-stage malaria antigens that had been fractionated by isoelectric focusing using a variety of Ampholines over the range pH 3 to 10. Fractions were tested for their ability to protect against live challenge and to prime for parasite-specific T-cell help. Both activities exhibited three major peaks in the pH regions 4.5, 6.5, and 8, the pH 4.5 peak being the most consistently protective. There was a significant correlation between protection and T-helper-cell priming, particularly with antigens from the first peak, suggesting that T-cell priming represents an important component of the function of some protective malaria vaccines.     

Immunization with a combination of merozoite surface proteins 4/5 and 1 enhances protection against lethal challenge with Plasmodium yoelii

It is widely believed that subunit vaccines composed of multiple components will offer greater protection against challenge by malaria, and yet there is little experimental evidence to support this view. We set out to test this proposition in the Plasmodium yoelii challenge system in rodents by comparing the degree of protection conferred by immunization with a mixture of merozoite surface proteins to that conferred by single proteins. We therefore examined a defined protein mixture made of the epidermal growth factor-like domains of P. yoelli merozoite surface protein 1 (MSP1) and MSP4/5, the homologue of P. falciparum MSP4 and MSP5. In the present study we demonstrate that this combination of recombinant proteins dramatically enhances protection against lethal malaria challenge compared to either protein administered alone. Many mice immunized with the MSP4/5 plus MSP1(19) combination did not develop detectable parasitemia after challenge. Combined immunization with MSP1(19) and yMSP4/5, a product characterized by lower protective efficacy, also greatly enhanced protection by reducing peak parasitemias and increasing the numbers of survivors. In some combination trials, levels of antibodies to MSP1(19) were elevated compared to the MSP1(19) alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP1(19) response was observed. Boosting of anti-MSP1(19) did not appear to be due to contaminating endotoxin in the EcMSP4/5 material since enhanced protection was observed in C3H/HeJ mice, which are endotoxin insensitive. Collectively, these experiments show that multiantigen combinations offer enhanced levels of protection against asexual stage infection and suggest that combinations of MSP1, MSP4, and MSP5 should be evaluated further for use in humans.     

Long-term flaxseed oil supplementation diet protects BALB/c mice against Streptococcus pneumoniae infection

Intense host immune response to infection contributes significantly to the pathology of pneumococcal pneumonia. Therefore, the regulation of host immune response is critical for the successful outcome of pneumonia in such patients. The aim of the present study was to investigate the effect of n-3 PUFA, i.e. flaxseed oil supplementation for short (4 weeks) as well as long (9 weeks) term, on the course of S. pneumoniae D39 serotype 2 infection in mice. The efficacy of flaxseed oil supplementation was investigated in terms of survival of animals and production of various inflammatory molecules (malondialdehyde, myeloperoxidase, nitric oxide) in the lung homogenate of animals. This was correlated with bacteriological and histopathological parameters. The immunomodulation was studied in terms of cytokines in the lungs following infection with Streptococcus pneumoniae. Results suggest that long-term flaxseed supplementation protected the mice against bacterial colonization of lungs with Streptococcus pneumoniae with reduced histopathological involvement of lung tissue. Moderate pneumonia was observed in supplemented, infected mice compared to severe pneumonia seen in control mice. This was accompanied by decreased inflammatory markers (malondialdehyde, myeloperoxidase, nitric oxide) as the disease progressed. In addition, difference in the levels of pro-inflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10) cytokines was observed in the flaxseed fed animals. On the contrary, short-term supplementation did not show such an effect on lung colonization.     

幽门螺杆菌感染小鼠及其抗原免疫小鼠后体液免疫应答的比较

目的：比较BALB／c小鼠感染H.pylori后以及经H.pylori UreB抗原口服免疫后的体液免疫应答的差异。方法：80只BALB／c小鼠分为感染组和免疫组，感染组灌喂H.pylori小鼠适应株；免疫组灌喂重组H.pylori UreB抗原和佐剂LTB。在试验的0，2，6和10周时每组分别取10只小鼠收集唾液及血液标本，用ELISA法检测抗UreBIgG和IgA抗体，同时采集胃组织作H.pylori感染检测。结果：感染组小鼠在灌喂H.pylori后2，6，10周，感染率均为100％，其血清中抗UreB IgG增高明显，但血清及唾液中均未见IgA的明显升高，；免疫组小鼠在初次免疫后第6，10周后，血清及唾液中抗UreB,IgG和IgA抗体的均显著升高。结论：H.yplori感染BALB／ c小鼠不能诱导其产生明显的特异性黏膜免疫应答，而重组UreB可作为良好的免疫原诱导其产生分泌型IgA抗体。     

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.     

A human antibody against anthrax protective antigen protects rabbits from lethal infection with aerosolized spores

ALXN4100, a fully human antibody that binds to the protective antigen of anthrax toxin, was generated from a Fab isolated from a phage display library and was analyzed for its pharmacokinetic properties in rabbits and then used to protect rabbits from challenge with a lethal aerosol dose of Bacillus anthracis spores (approximately 322X LD(50)). All rabbits receiving 15 or 40 mg/kg of antibody 24 hours before challenge survived; survival of rabbits receiving 4 mg/kg either subcutaneously or intravenously was 80 or 90%, respectively. Susceptibility to anthrax disease appeared to be correlated with serum antibody concentration. This study indicates that ALXN4100 has the potential to be useful for prophylaxis of anthrax disease in humans.     

An anti-CD3 monoclonal antibody protects mice against a lethal infection with Listeria monocytogenes through induction of endogenous cytokines.

Mice were protected against a lethal infection with Listeria monocytogenes when treated with low doses of an anti-CD3 monoclonal antibody (MAb). Injection of anti-CD3 MAb induced rapid production of endogenous tumor necrosis factor (TNF) in the spleens and endogenous gamma interferon (IFN-gamma) in the bloodstreams and spleens of mice. Administration of anti-Thy1.2 MAb or a combination of anti-CD4 MAb and anti-CD8 MAb resulted in suppression of anti-CD3 MAb-induced endogenous cytokine production and antilisterial resistance. Alternatively, in vivo depletion of anti-CD3 MAb-induced TNF and IFN-gamma by the simultaneous administration of antibodies against TNF and IFN-gamma suppressed anti-CD3 MAb-induced antilisterial resistance. Moreover, injection of anti-complement receptor type 3 (Mac-1, CD11b) resulted in inhibition of anti-CD3 MAb-induced antilisterial resistance. These results suggest that the preventive effect of anti-CD3 MAb might be due to activation of phagocytes by TNF and IFN-gamma induced by stimulating CD4+ T cells and CD8+ T cells with the MAb. Furthermore, treatment with anti-CD3 MAb did not inhibit establishment of acquired resistance against secondary infection with L. monocytogenes.     

Oral immunization with a combination of Plasmodium yoelii merozoite surface proteins 1 and 4/5 enhances protection against lethal malaria challenge

Oral immunization of mice with Escherichia coli-expressed Plasmodium yoelii merozoite surface protein 4/5 or the C-terminal 19-kDa fragment of merozoite surface protein 1 induced systemic antibody responses and protected mice against lethal malaria infection. A combination of these two proteins administered orally conferred improved protection compared to that conferred by either protein administered alone.     

Passive immunization of chickens against Eimeria maxima infection with a monoclonal antibody developed against a gametocyte antigen

Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.     

Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies secreted by six hybridomas and recognizing antigenic sites on glycoproteins gC, gAB, gD, gE, and gF of herpes simplex virus type 2 were examined for their ability to protect BALB/c mice from lethal infection by the virus. Administration of monoclonal antibodies to individual glycoproteins intraperitoneally 3 h before footpad challenge with 10 times the 50% lethal dose of virus protected between 35 and 75% of the mice, except for one of two monoclonal antibodies recognizing antigens on gC. The antibodies did not neutralize virus in vitro and protected A/J mice deficient in the fifth component of complement as efficiently as complement-sufficient BALB/c mice. A good correlation was found between protection and titers of monoclonal antibodies assessed by antibody-dependent cell-mediated cytolysis. The results indicate that any of the glycoproteins can serve as antigens for a protective immune response. In addition, the data are compatible with protection being mediated by an antibody-dependent cell-mediated cytolysis mechanism.     

Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4

Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of underlying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1). Immunologically intact or selected immunologic knockout mice were immunized with purified recombinant Plasmodium chabaudi AMA-1 (PcAMA-1) and/or the 42-kDa C-terminal processing fragment of P. chabaudi MSP-1 (MSP-1(42)). The efficacy of immunization in each animal model was measured as protection against blood-stage P. chabaudi malaria. Immunization of B-cell-deficient JH(-/-) mice indicated that PcAMA-1 vaccine-induced immunity is largely antibody dependent. In contrast, JH(-/-) mice immunized with PcMSP-1(42) were partially protected against P. chabaudi malaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of gammadelta T cells in vaccine-induced PcAMA-1 and/or PcMSP-1(42) protection was minor. Analysis of the isotypic profile of antigen-specific antibodies induced by immunization of immunologically intact mice revealed a dominant IgG1 response. However, neither interleukin-4 and the production of IgG1 antibodies nor gamma interferon and the production of IgG2a/c antibodies were essential for PcAMA-1 and/or PcMSP-1(42) vaccine-induced protection. Therefore, for protective antibody-mediated immunity, vaccine adjuvants and delivery systems for AMA-1- and MSP-1-based vaccines can be selected for their ability to maximize responses irrespective of IgG isotype or any Th1 versus Th2 bias in the CD4(+)-T-cell response.     

Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.