Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement

C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes.     

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Immunostimulatory DNA-based vaccines elicit multifaceted immune responses against HIV at systemic and mucosal sites

Immunostimulatory DNA sequences (ISS, also known as CpG motifs) are pathogen-associated molecular patterns that are potent stimulators of innate immunity. We tested the ability of ISS to act as an immunostimulatory pathogen-associated molecular pattern in a model HIV vaccine using gp120 envelope protein as the Ag. Mice immunized with gp120 and ISS, or a gp120:ISS conjugate, developed gp120-specific immune responses which included: 1) Ab production; 2) a Th1-biased cytokine response; 3) the secretion of beta-chemokines, which are known to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5) mucosal immune responses; and 6) CD8 T cell responses that were independent of CD4 T cell help. Based on these results, ISS-based immunization holds promise for the development of an effective preventive and therapeutic HIV vaccine.     

Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.     

Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice

Group B Streptococcus (GBS) is the foremost bacterial cause of serious neonatal infections. Protective immunity to GBS is mediated by specific Abs to the organism's capsular polysaccharide Ags. To examine the role of complement in the humoral immune response to type III GBS capsular polysaccharide (III-PS), mice deficient in C3 or in CD21/CD35 (i.e., complement receptors 1 and 2; CR1/CR2) were immunized with III-PS. Mice deficient in C3 or Cr2 had an impaired primary immune response to III-PS. The defective response was characterized by low IgM levels and the lack of an isotype switch from IgM to IgG Ab production. Compared with wild-type mice, C3- and Cr2-deficient mice exhibited decreased uptake of III-PS by follicular dendritic cells within the germinal centers and impaired localization of III-PS to the marginal zone B cells. Complement-dependent uptake of capsular polysaccharide by marginal zone B cells appears necessary for an effective immune response to III-PS. The normal immune response in wild-type mice may require localization of polysaccharide to marginal zone B cells with subsequent transfer of the Ag to follicular dendritic cells.     

Evidence for early aging in the mucosal immune system

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 microg of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21. An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level. When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4(+) T cells from spleen and Peyer's patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s. c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice. These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.     

Ag85B of mycobacteria elicits effective CTL responses through activation of robust Th1 immunity as a novel adjuvant in DNA vaccine

CD4+ T cells play a crucial role in CTL generation in a DNA vaccination strategy. Several studies have demonstrated the requirement of CD4+ T cells for the induction of a sufficient immune response by coadministrating DNAs. In the present study we investigated the effectiveness of Ag85B of mycobacteria, which is known to be one of the immunogenic proteins for Th1 development, as an adjuvant of a DNA vaccine. HIV gp120 DNA vaccine mixed with Ag85B DNA as an adjuvant induced HIV gp120-specific Th1 responses, as shown by delayed-type hypersensitivity, cytokine secretion, and increasing HIV-specific CTL responses. Moreover, these responses were enhanced in mice primed with Mycobacterium bovis bacillus Calmette-Guérin before immunization of HIV DNA vaccine mixed with Ag85B DNA. Furthermore, these immunized mice showed substantial reduction of HIV gp120-expressing recombinant vaccinia virus titers compared with the titers in other experimental mice after recombinant vaccinia virus challenge. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing CTL responses in a DNA vaccination strategy.     

HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors

Switching from IgM to IgG and IgA is essential for antiviral immunity and requires engagement of CD40 on B cells by CD40L on CD4(+) T cells. HIV-1 is thought to impair CD40-dependent production of protective IgG and IgA by inducing progressive loss of CD4(+) T cells. Paradoxically, this humoral immunodeficiency is associated with B cell hyperactivation and increased production of nonprotective IgG and IgA that are either nonspecific or specific for HIV-1 envelope glycoproteins, including gp120. Nonspecific and gp120-specific IgG and IgA are sensitive to antiretroviral therapy and remain sustained in infected individuals with very few CD4(+) T cells. One interpretation is that some HIV-1 Ags elicit IgG and IgA class switch DNA recombination (CSR) in a CD40-independent fashion. We show that a subset of B cells binds gp120 through mannose C-type lectin receptors (MCLRs). In the presence of gp120, MCLR-expressing B cells up-regulate the CSR-inducing enzyme, activation-induced cytidine deaminase, and undergo CSR from IgM to IgG and IgA. CSR is further enhanced by IL-4 or IL-10, whereas Ab secretion requires a B cell-activating factor of the TNF family. This CD40L-related molecule is produced by monocytes upon CD4, CCR5, and CXCR4 engagement by gp120 and cooperates with IL-4 and IL-10 to up-regulate MCLRs on B cells. Thus, gp120 may elicit polyclonal IgG and IgA responses by linking the innate and adaptive immune systems through the B cell-activating factor of the TNF family. Chronic activation of B cells through this CD40-independent pathway could impair protective T cell-dependent Ab responses by inducing immune exhaustion.     

CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154-/-) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154-/- mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154-/- mice exhibited a defect both in the production of IL-12, IFN-gamma, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154-/- animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in > or =90% protection. The lack of protective immunity in CD154-/- mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154-/- mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.     

Long-term maintenance of gp120-specific immune responses by genetic vaccination with the HIV-1 envelope genes linked to the gene encoding Flt-3 ligand

DNA vaccines target dendritic cells (DC) to induce Ag-specific immune responses in animals. Potent HIV-specific immunity could be achieved by efficient priming of the immune system by DNA vaccines. We investigated a novel DNA vaccine approach based on the role of growth factors in DC expansion and differentiation. To this end, we constructed chimeric genes encoding the HIV envelope glycoproteins physically linked to the extracellular domain of Fms-like tyrosine kinase receptor-3 ligand (FLex; a DC growth factor; both mouse (m)FLex and human (h)FLex). These chimeric gene constructs synthesized biologically active, oligomeric FLex:gp120 fusion proteins and induced DC expansion (CD11c(+)CD11b(+)) when injected i.v. into mice. This DC expansion is comparable to that achieved by FLex DNA encoding native FLex protein. When delivered intramuscularly as DNA vaccines, hFLex:gp120 induced high frequencies of gp120-specific CD8(+) T cells in the presence or absence of FLex DNA-induced DC expansion, but gp120 and mFLex:gp120 elicited only low to moderate levels of Ag-specific CD8(+) T cells. In contrast, mFLex:gp120 induced high levels of anti-gp120 Abs under identical conditions of DNA vaccination. However, the Ab levels in mice immunized with DNA vaccines encoding hFLex:gp120 and gp120 proteins were low without DC expansion, but reached high levels comparable to that elicited by mFLex:gp120 only after the second boost in the presence of DC expansion. Importantly, the gp120-specific CD8(+) T cells persisted at high frequency for 114 days (16 wk) after a booster injection. These experiments provide insight into the importance of modulating DC function in vivo for effective genetic vaccination in animals.     

A novel adjuvant for mucosal immunity to HIV-1 gp120 in nonhuman primates

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4(+)) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.     

Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19(-/-) and CD21(-/-) mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.     

Cross-priming as a predominant mechanism for inducing CD8(+) T cell responses in gene gun DNA immunization.

DNA immunization induces CD8(+) CTL responses by bone marrow-derived APCs, which are directly transfected with a plasmid DNA and/or acquire Ags from DNA-transfected non-APCs. To investigate the relative contribution of DNA-transfected APCs vs non-APCs to the initiation of CD8(+) T cell responses, we used tissue-specific promoter-directed gene expression and adoptive transfer systems in gene gun DNA immunization. In this study, we demonstrated that non-APC-specific gene expressions induced significant CD8(+) CTL and IFN-gamma-producing cells and Ab responses, whereas APC-specific gene expressions led to moderate CTL and IFN-gamma-producers, but no Ab responses. Interestingly, mice immunized with a non-APC-specific plasmid induced more rapid, vigorous, and prolonged proliferation of adoptively transferred Ag-specific CD8(+) T cells than APC-specific plasmid-immunized mice. In addition, the in vivo proliferative responses elicited by a non-APC-specific plasmid administration were dependent on TAP, but were independent of CD4(+) T cell help. Collectively, our results suggest that cross-priming, in which Ags expressed in non-APCs are taken up, processed, and presented by APCs, plays an important role in the initiation, magnitude, and maintenance of CD8(+) T cell responses in gene gun DNA immunization.     

HIV mucosal vaccine: nasal immunization with gp160-encapsulated hemagglutinating virus of Japan-liposome induces antigen-specific CTLs and neutralizing antibody responses

Nasal immunization of normal mice with HIVgp160-encapsulated hemagglutinating virus of Japan (HVJ)-liposome induced high titers of gp160-specific neutralizing IgG in serum and IgA in nasal wash, saliva, fecal extract, and vaginal wash, along with both Th1- and Th2-type responses. HIVgp160-specific IgG- and IgA-producing cells were also detected in mononuclear cells isolated from spleen, nasal cavity, salivary gland, intestinal lamina propria, and vaginal tissue of nasally immunized mice. In addition, CD8(+) CTLs were induced in mice nasally immunized with gp160-HVJ-liposome. These findings suggest that two layers of effective HIV-specific humoral and cellular immunity, in mucosal and systemic sites, were induced by this nasal vaccine. In immunodeficient mice, nasal immunization with gp160-HVJ-liposome induced Ag-specific immune responses for the systemic and mucosal compartments of both Th1 (IFN-gamma(-/-)) and Th2 (IL-4(-/-)). In vitro Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and IgG Abs that had been induced by nasal immunization with gp160-HVJ-liposome were able to neutralize a clinically isolated strain of HIV-MN strain isolated from Japanese hemophiliac patients. Taken together, these results suggest that, for the prevention and control of AIDS, nasally administered gp160-HVJ-liposome is a powerful immunization tool that induces necessary Ag-specific immune responses at different stages of HIV infection.     

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.     

Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.     

Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.