Vaccination of mice with a 30 kDa Schistosoma antigen with and without human adjuvant induces high protection against S. mansoni infection

A 30 kDa antigen was characterized as a hydrophobic polypeptide containing 16 amino acids and evaluated as a potential candidate vaccine against infection by Schistosoma mansoni. CD1 albino mice immunized at 0, 14, and 21 days with 25 or 50 microg of the 30 kDa antigen per mouse with and without alum developed high levels of IgG antibodies (predominantly IgG2a and IgG2b isotypes). When immunized mice were infected with 200 S. mansoni cercariae, the highest protection levels (61% and 65% reduction in worm burden in two separate experiments) were obtained using the 50-microg antigen without alum adjuvant. The granuloma size decreased to 10%, a non-significant level in mice immunized using alum adjuvant. The results demonstrate the ability of the 30 kDa antigen with and without alum adjuvant to protect mice against S. mansoni infection.     

Influence of antibody isotype on passive serotherapy of lymphoma

We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.     

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.     

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.     

A viral vectored prime-boost immunization regime targeting the malaria Pfs25 antigen induces transmission-blocking activity

The ookinete surface protein Pfs25 is a macrogamete-to-ookinete/ookinete stage antigen of Plasmodium falciparum, capable of exerting high-level anti-malarial transmission-blocking activity following immunization with recombinant protein-in-adjuvant formulations. Here, this antigen was expressed in recombinant chimpanzee adenovirus 63 (ChAd63), human adenovirus serotype 5 (AdHu5) and modified vaccinia virus Ankara (MVA) viral vectored vaccines. Two immunizations were administered to mice in a heterologous prime-boost regime. Immunization of mice with AdHu5 Pfs25 at week 0 and MVA Pfs25 at week 10 (Ad-MVA Pfs25) resulted in high anti-Pfs25 IgG titers, consisting of predominantly isotypes IgG1 and IgG2a. A single priming immunization with ChAd63 Pfs25 was as effective as AdHu5 Pfs25 with respect to ELISA titers at 8 weeks post-immunization. Sera from Ad-MVA Pfs25 immunized mice inhibited the transmission of P. falciparum to the mosquito both ex vivo and in vivo. In a standard membrane-feeding assay using NF54 strain P. falciparum, oocyst intensity in Anopheles stephensi mosquitoes was significantly reduced in an IgG concentration-dependent manner when compared to control feeds (96% reduction of intensity, 78% reduction in prevalence at a 1 in 5 dilution of sera). In addition, an in vivo transmission-blocking effect was also demonstrated by direct feeding of immunized mice infected with Pfs25DR3, a chimeric P. berghei line expressing Pfs25 in place of endogenous Pbs25. In this assay the density of Pfs25DR3 oocysts was significantly reduced when mosquitoes were fed on vaccinated as compared to control mice (67% reduction of intensity, 28% reduction in prevalence) and specific IgG titer correlated with efficacy. These data confirm the utility of the adenovirus-MVA vaccine platform for the induction of antibodies with transmission-blocking activity, and support the continued development of this alternative approach to transmission-blocking malaria subunit vaccines.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.     

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <. 001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.     

Oral-tolerance induction in diet-induced obese mice

OBJECTIVE: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. RESEARCH METHODS AND PROCEDURES: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. RESULTS: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. DISCUSSION: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.     

TNP-LPS induces an IgG anti-TNP immune response in mice.

The trinitrophenylated derivatives of lipopolysaccharide (TNP-LPS) elicit a specific anti-TNP, thymus-independent immune response in mice. After a single injection of antigen, anti-TNP antibodies of IgM and IgG isotypes are detected at the cellular and at the humoral levels, in athymic nude mice as well as in conventional (C57B1/6 × DBA/2)F₁ mice. The immune sera were resolved into IgM and IgG molecules by gel filtration; both fractions showed an anti-TNP activity, thus confirming the data obtained by the cellular analysis.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Induction of systemic Th1 and Th2 immune responses by oral administration of soluble antigen and diesel exhaust particles

The present study was undertaken to examine whether oral administration of soluble antigen together with diesel exhaust particles (DEP) induced the systemic immune response in mice. Mice were orally given 1 mg of hen egg lysozyme (HEL) with varying doses of DEP every 3 days over a period of 15 days. The results showed that oral administration of HEL plus DEP produced anti-HEL IgG antibodies in serum in a dose-related fashion, while either HEL or DEP alone failed to show the antigen-specific IgG antibody production. Production of anti-HEL IgG2a and IgG1 antibodies, which are dependent on Th1 and Th2 CD4(+) T cells, respectively, was seen in mice fed with combined HEL and DEP, although anti-HEL IgG1 antibodies appeared to be more efficiently produced by lower doses of DEP than anti-HEL IgG2a antibodies. There was marked antigen-specific proliferation of spleen cells in mice treated with HEL and DEP. The anti-HEL antibody production and lymphoid cell proliferation to the antigen were associated with marked secretion of the Th1 cytokine IFN-gamma as well as the Th2 cytokine IL-4. These results suggest that DEP may act as a mucosal adjuvant in the gut enhancing systemic Th1 and Th2 immune responses and might play a role in oral immunization and food allergy.     

The in vivo effects of neutralizing antibodies against IFN-γ, IL-4, or IL-10 on the humoral immune response in young and aged mice

In the present study we investigated whether age-related changes in the composition and functional properties of murine CD4⁺ T cells are reflected in vivo by a changed humoral response to influenza vaccine in aged mice. After the primary immunization, the titers of influenza-specific IgM, IgG1, IgG2a, and IgG2b, but not of IgG3 and IgE, were significantly reduced in aged mice compared to young mice. Treatment of aged mice with anti-IFN-γ, anti-IL-4, or anti-IL-10 resulted in levels of IgM and IgG1 comparable to those found in young mice, whereas IgG2a and IgG2b were further decreased. After the booster immunization IgE was significantly enhanced in aged mice, whereas no differences were observed with regard to the other isotypes. During the primary response in young mice, anti-IFN-γ stimulated IgG1 and IgE, whereas an inhibition of IgG2a, IgG2b, and IgG3 was observed. Anti-IL-4 caused a decrease only in IgG3 while anti-IL-10 increased IgM and IgG1 and decreased IgG2b and IgG3. During the primary response in aged mice, all anti-cytokine antibodies enhanced IgM and IgG1 while IgE was only enhanced by anti-IL-10. By contrast, IgG3 was inhibited by anti-IFN-γ and anti-IL-10. Anti-cytokine treatment of young mice increased all isotypes, except IgG3, in the secondary response, whereas the secondary response in aged mice was largely insensitive to anti-cytokine treatment. These data therefore support the idea that the in vivo effects of cytokines on isotype switching are dependent on the differentiation stage of B cells which may be different in young and aged mice.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.     

Rapid anti-helminthic response of B lymphocytes in the intestinal mucosal tissues of rats

B cell response to Trichinella spiralis (Ts) adult antigen (Ag) was studied in rats 1-20 days postinfection. B cell recoveries from the mesenteric lymph node (MLN), Peyer's patches (PP), thoracic duct lymph (TDL), and the spleen were determined by FACS analysis and Ag-specific antibody-producing cells (Ab-pc) in these tissues were enumerated using the immunoplaque assay. Total B cell numbers increased 2-70 times from day 3 postinfection in the MLN and TDL obtained from MLN-resected rats (MX) and such proliferation was not found in the PP or the spleen. Ab-pc of all isotypes increased from day 3 in the MLN and from day 2 in the MX-TDL. Among all isotypes, IgE- and IgG1-pc showed the strongest response. Immunofluorescence study revealed that these B cells were activated in the non-PP region of the small intestine. These results indicate an early isotype switch to IgG1 and IgE production in Ts-infected small intestine.     

DNA immunization with Plasmodium falciparum serine repeat antigen: regulation of humoral immune response by coinoculation of cytokine expression plasmid

We immunized mice with plasmid expressing the 47-kDa amino-terminal domain of the Plasmodium falciparum serine repeat antigen (SERA) using gene gun and investigated humoral immune response to SERA antigen. Significant SERA-specific IgG was observed in BALB/c mice after immunization three times with SERA expression plasmid. Furthermore, these levels were increased by the coinoculation of cytokine (IFN-gamma, IL-4, GM-CSF, or IL-12) expression plasmid. In respect to the SERA-specific Ig subclasses, coinoculation of IFN-gamma, GM-CSF, or IL-12 expression plasmid increased the levels of SERA-specific IgG2a, and these were much higher than that in mice immunized with SERA expression plasmid alone. In contrast to the SERA-specific IgG2a, coinoculation of any cytokine expression plasmid did not change the levels of SERA-specific IgG1. These results indicate that cytokine expression plasmid enhances and regulates humoral immune response elicited by SERA DNA immunization.     

Linkage of CD40L to a self-tumor antigen enhances the antitumor immune responses of dendritic cell-based treatment

CD40-CD40 ligand (CD40L) interaction is an important costimulatory signal in the interaction between T cells and antigen-presenting cells (APC). In the present study, we determined whether the linkage of CD40L to the tumor-specific idiotype (Id) derived from a murine B-cell lymphoma, 38C13, could enhance its immunogenicity when presented by dendritic cells (DC). We showed that bone marrow-derived DC pulsed with Id-CD40L upregulated the expression of CD40, CD80, CD86, and major histocompatibility complex (MHC) class II molecules with the increased production of interleukin-12 (IL-12). Mice immunized with DC loaded with Id-CD40L showed high levels of anti-Id antibody response of both IgG2a and IgG1 isotypes. In addition, nylon wool-enriched T cells from these immunized mice showed a tumor-specific T-cell proliferative response upon stimulation with Id protein. Mice immunized with DC pulsed with Id alone failed to show any of these immune responses. Immunization with DC pulsed with Id-CD40L showed increased resistance to the challenge by 38C13 tumor, and tumor growth was significantly retarded. Together, these results show that linkage of CD40L to a self-tumor antigen enhances the anti-tumor immune response in DC-based treatment.     

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.     

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni²⁺ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.