Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Priming of human papillomavirus type 11-specific humoral and cellular immune responses in college-aged women with a virus-like particle vaccine

In this study, we evaluated the potency of a human papillomavirus (HPV) virus-like particle (VLP)-based vaccine at generating HPV type 11 (HPV-11)-specific cellular and humoral immune responses in seronegative women. The vaccine was administered by intramuscular immunizations at months 0, 2, and 6. A fourth immunization was administered to approximately half of the women at month 12. All vaccine recipients had positive HPV-11 VLP-specific lymphoproliferative responses at month 3 following the second immunization (geometric mean lymphoproliferative stimulation index [SI] = 28.4; 95% confidence interval [CI] = 16.9 to 48.0) and HPV-11 VLP-specific antibody titers following the first immunization at month 1 (geometric mean antibody titer = 53.9 milli-Merck units/ml, 95% CI, 34.8 to 83.7). In contrast, lymphoproliferative and antibody titer responses were never detected in the participants who received placebo. Relatively homogeneous lymphoproliferative responses were observed in all vaccinated women. The mean lymphoproliferative SI of the vaccinated group over the first 12 months of the study was 7.6-fold greater than that of the placebo group following the initial immunization. The cellular immune responses generated by VLP immunization were both Th1 and Th2, since peripheral blood mononuclear cells from vaccinees, but not placebo recipients, secreted interleukin 2 (IL-2), IL-5, and gamma interferon (IFN-gamma) in response to in vitro stimulation with HPV-11 VLP. The proliferation-based SI was moderately correlated with IFN-gamma production and significantly correlated with IL-2 production after the third immunization (P = 0.078 and 0.002, respectively). The robust lymphoproliferative responses were specific for HPV-11, since SIs generated against bovine papillomavirus and HPV-16 VLPs were not generally observed and when detected were similar pre- and postimmunization.     

Pseudovirion particle production by live poxvirus human immunodeficiency virus vaccine vector enhances humoral and cellular immune responses

Live-vector-based human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation. Live vectors that carry an intact gag gene are capable of eliciting HIV pseudovirion particle formation from infected host cells. The impact of pseudovirion particle formation on the immune response generated by live HIV vaccine vectors has not been established. In this study, a canarypox HIV vaccine candidate vector expressing HIV gag and env genes, vCP205, was modified by the introduction of a glycine-to-alanine coding change in the N-terminal myristylation site of gag to create Myr- vCP205. This substitution effectively eliminated particle formation without altering the level of protein production. vCP205 and Myr- vCP205 were then directly compared for the ability to induce HIV-specific immune responses in mice. The particle-competent vector vCP205 elicited higher levels of CD8+ T-cell responses, as indicated by gamma interferon enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining. Humoral responses to Gag and Env were also markedly higher from animals immunized with the particle-competent vector. Furthermore, HIV-specific CD4+ T-cell responses were greater among animals immunized with the particle-competent vector. Using a human dendritic cell model of antigen presentation in vitro, vCP205 generated greater ELISPOT responses than Myr- vCP205. These results demonstrate that pseudovirion particle production by live-vector HIV vaccines enhances HIV-specific cellular and humoral immune responses.     

Targeting with bovine CD154 enhances humoral immune responses induced by a DNA vaccine in sheep

CD40-CD154 interactions play an important role in regulating humoral and cell-mediated immune responses. Recently, these interactions have been exploited for the development of therapeutic and preventive treatments. The objective of this study was to test the ability of bovine CD154 to target a plasmid-encoded Ag to CD40-expressing APCs. To achieve this, a plasmid coding for bovine CD154 fused to a truncated secreted form of bovine herpesvirus 1 glycoprotein D (tgD), pSLIAtgD-CD154, was constructed. The chimeric tgD-CD154 was expressed in vitro in COS-7 cells and reacted with both glycoprotein D- and CD154-specific Abs. Both tgD and tgD-CD154 were capable of binding to epithelial cells, whereas only tgD-CD154 bound to B cells. Furthermore, dual-labeling of ovine PBMCs revealed that tgD-CD154 was bound by primarily B cells. The functional integrity of the tgD-CD154 chimera was confirmed by the induction of both IL-4-dependent B cell proliferation and tgD-specific lymphoproliferative responses in vitro. Finally, sheep immunized with pSLIAtgD-CD154 developed a more rapid primary tgD-specific Ab response and a significantly stronger tgD-specific secondary response when compared with animals immunized with pSLIAtgD and control animals. Similarly, virus-neutralizing Ab titers were significantly higher after secondary immunization with pSLIAtgD-CD154. These results demonstrate that using CD154 to target plasmid-expressed Ag can significantly enhance immune responses induced by a DNA vaccine.     

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.     

Immunostimulant adjuvant patch enhances humoral and cellular immune responses to DNA immunization

The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches.     

Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon(+) (IFN-gamma(+))CD4(+) and IFN-gamma(+)CD8(+) T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.     

Longitudinal analysis of human humoral responses after vaccination with a live attenuated V. cholerae vaccine

Vibrio cholerae is a bacterial pathogen which causes the severe acute diarrheal disease cholera. Given that a symptomatic incident of cholera can lead to long term protection, a thorough understanding of the immune response to this pathogen is needed to identify parameters critical to the generation and durability of immunity. To approach this, we utilized a live attenuated cholera vaccine to model the response to V. cholerae infection in 12 naïve subjects. We found that this live attenuated vaccine induced durable vibriocidal antibody titers that were maintained at least one year after vaccination. Similar to what we previously reported in infected patients from Bangladesh, we found that vaccination induced plasmablast responses were primarily specific to the two immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). Interestingly, the magnitude of the early plasmablast response at day 7 predicted the serological outcome of vaccination at day 30. However, this correlation was no longer present at later timepoints. The acute responses displayed preferential immunoglobulin isotype usage, with LPS specific cells being largely IgM or IgA producing, while cholera toxin responses were predominantly IgG. Finally, CCR9 was highly expressed on vaccine induced plasmablasts, especially on IgM and IgA producing cells, suggesting a role in migration to the gastrointestinal tract. Collectively, these findings demonstrate that the use of a live attenuated cholera vaccine is an effective tool to examine the primary and long-term immune response following V. cholerae exposure. Additionally, it provides insight into the phenotype and specificity of the cells which likely return to and mediate immunity at the intestinal mucosa. A thorough understanding of these properties both in peripheral blood and in the intestinal mucosae will inform future vaccine development against both cholera and other mucosal pathogens.Trial Registration:{"type":"clinical-trial","attrs":{"text":"NCT03251495","term_id":"NCT03251495"}}NCT03251495.     

Th cell-independent immune responses to chimeric hemagglutinin/simian human immunodeficiency virus-like particles vaccine

CD4(+) Th cells are believed to be essential for the induction of humoral and cellular immune responses. In this study we tested the effect and possible mechanisms of the major antigenic component in influenza, hemagglutinin (HA), in helping HIV Env to induce immune responses in CD4(+) T cell knockout (CD4 KO) mice. Simian HIV virus-like particles (SHIV VLPs) or phenotypically mixed chimeric influenza HA/SHIV VLPs were used as immunogens to immunize CD4 KO mice either i.p. or intranasally (i.n.). We found that chimeric HA/SHIV VLPs significantly induced a greater IgG Ab response in both i.p. and i.n. immunized mice and a greater IgA Ab response in mucosal washes in i.n. immunized mice compared with SHIV VLPs. Importantly, chimeric HA/SHIV VLPs induced approximately 3-fold higher neutralizing Ab titers against HIV 89.6 than SHIV VLPs in the absence of CD4(+) T cell help. There was also approximately 40% more specific lysis of the HIV Env-expressing target cells in chimeric HA/SHIV VLP-immunized than in SHIV VLP-immunized CD4 KO mouse splenocytes. Moreover, we have found that chimeric HA/SHIV VLPs could efficiently bind and activate dendritic cells and stimulate the activated dendritic cells to secret TNF-alpha and IFN-gamma. Therefore, chimeric HA/SHIV VLPs could efficiently prime and activate APCs, which could, in turn, induce immune responses in a CD4(+) T cell-independent manner. This study suggests a novel adjuvant role of influenza HA as well as a new strategy to develop more effective therapeutic vaccines for AIDS patients with low CD4(+) T cell counts.     

Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination

Background

The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis.

Methods and Principal Findings

In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study.

Conclusion and Significance

Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.     

Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses

Peyer's patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA(+) and IgG2a(+) B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.     

Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Increasing M2 epitope density enhances systemic and mucosal immune responses to influenza A virus

Recombinant proteins bearing one (sM2) or three (3sM2) copies of M2 epitope from influenza virus were expressed in Escherichia coli. Mice were administrated with these two proteins and systemic and mucosal immune responses were analyzed. Compared with sM2, 3sM2 induced M2-specific serum IgG and mucosal IgA antibodies with neutralizing activity more efficiently in the whole immune course and the later mucosal immunization improved this advantage. MDCK cells pretreated with 3sM2 antisera showed less pathogenic morphological changes compared with that of pretreated with sM2. Together, our results demonstrated that high epitope density in one recombinant protein can enhance humoral immune responses in both systemic and mucosal immunization, and combination of systemic and mucosal immunization enhances this advantage.     

滴鼻免疫有效的诱导粘膜及系统免疫反应

为了探讨粘膜疫苗滴鼻免疫对小鼠不同粘膜部位和系统免疫部位的影响,将BALB/c小鼠随机分为三组,每组15只,FSM-2117或FS-5416 4×107cfu/只经滴鼻途径免疫小鼠,间隔两周,4次免后7天活杀,收集鼻咽、肺、肠、生殖道冲洗液和血清,ELISA法检测其中特异性抗福氏、宋内氏LPSIg和AIgG;分离NALT、鼻通道、脾、小肠PP淋巴细胞,细胞中加入全菌破碎抗原共培养,于不同时间取出细胞,提取RNA做RT-PCR.两株菌苗经鼻内免疫均诱发了鼻咽、肺、胃肠道和生殖道等不同粘膜部位及血清中特异性抗福氏、宋内氏LPSIgA、IgG的显著增加(P＜0.01);免疫动物的NALT、NP、PP结淋巴细胞在体外经抗原刺激后均在不同时间出现IFN-γ和IL-4mRNA的表达,而TGF-β mRNA的表达在免疫前后无明显变化.疫苗经鼻粘膜免疫可诱导不同粘膜部位及系统免疫反应的发生,鼻粘膜是一个安全有效的免疫途径.     

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.     

Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses

We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H(3)N(2)) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.