Intramuscular DNA immunization with in vivo electroporation induces antigen-specific cellular and humoral immune responses in both systemic and gut-mucosal compartments

Mucosal delivery of antigens induces antigen-specific immune responses in both systemic and mucosal compartments, and is an attractive approach for preventing initial infection with mucosal pathogens. It has been shown that the intramuscular (i.m.) immunization of plasmid DNA by in vivo electroporation (DNA e.p.) induces both cellular and humoral immune responses in the airway-mucosal compartment as well as in the systemic compartment, implying there is a mechanism that bridges between the systemic and mucosal immune responses. An important question is whether the i.m. DNA e.p.-immunization alone can induce antigen-specific immune responses in the gut-mucosal compartment. Here, we investigated the induction of antigen-specific CD8⁺ T cells and antibodies in both systemic and gut-mucosal compartments following i.m. DNA e.p.-immunization to mice. Surprisingly, the i.m. DNA e.p.-immunization induced the antigen-specific CD8⁺ T cells and antigen-specific antibodies in the gut-mucosal as well as the systemic compartment. These results suggest that the i.m. DNA e.p.-immunization should be considered as an effective vaccine strategy for the prevention of gut-mucosal infectious diseases.     

Recombinant rubella vectors elicit SIV Gag-specific T cell responses with cytotoxic potential in rhesus macaques

Live-attenuated rubella vaccine strain RA27/3 has been demonstrated to be safe and immunogenic in millions of children. The vaccine strain was used to insert SIV gag sequences and the resulting rubella vectors were tested in rhesus macaques alone and together with SIV gag DNA in different vaccine prime-boost combinations. We previously reported that such rubella vectors induce robust and durable SIV-specific humoral immune responses in macaques. Here, we report that recombinant rubella vectors elicit robust de novo SIV-specific cellular immune responses detectable for >10 months even after a single vaccination. The antigen-specific responses induced by the rubella vector include central and effector memory CD4⁺ and CD8⁺ T cells with cytotoxic potential. Rubella vectors can be administered repeatedly even after vaccination with the rubella vaccine strain RA27/3. Vaccine regimens including rubella vector and SIV gag DNA in different prime-boost combinations resulted in robust long-lasting cellular responses with significant increase of cellular responses upon boost. Rubella vectors provide a potent platform for inducing HIV-specific immunity that can be combined with DNA in a prime-boost regimen to elicit durable cellular immunity.     

Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7

Although many clinical trials on human papillomavirus (HPV) therapeutic vaccines have been performed, clinical responses have not been consistent. We have addressed mucosal cytotoxic cellular immune responses to HPV16 E7 after oral immunization of mice with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7). C57BL/6 mice were orally exposed to 0.1–100 mg/head of attenuated LacE7 or vehicle (Lac) vaccines at weeks 1, 2, 4, and 8. Responses to subcutaneous or intramuscular injection of an HPV16 E7 fusion protein using the same timing protocol were used for comparison. Oral immunization with LacE7 elicited E7-specific IFNγ-producing cells (T cells with E7-type1 immune responses) among integrin α4β7⁺ mucosal lymphocytes collected from gut mucosa. An induction of E7-specific granzyme B-producing cells (E7-CTL) exhibiting killer responses toward HPV16 E7-positive cells was also observed. The induction of T cells with specific mucosal E7-type1 immune responses was greater after oral immunization with LacE7 when compared to subcutaneous or intramuscular antigen delivery. Oral immunization with Lactobacillus-based vaccines was also able to induce mucosal cytotoxic cellular immune responses. This novel approach at a therapeutic HPV vaccine may achieve more effective clinical responses through its induction of mucosal E7-specific CTL.     

Comparative analysis of the immunogenicity of SARS-CoV nucleocapsid DNA vaccine administrated with different routes in mouse model

The development of strategies to augment the immunogenicity of DNA vaccines is critical for improving their clinical utility. One such strategy involves using the different immune routes with DNA vaccines. In the present study, the immunogenicity of SARS-CoV nucleocapsid DNA vaccine, induced by using the current routine vaccination routes (intramuscularly, by electroporation, or orally using live-attenuated Salmonella typhimurium), was compared in mouse model. The comparison between the three vaccination routes indicated that immunization intramuscularly induced a moderate T cell response and antibody response. Mice administrated by electroporation induced the highest antibody response among the three immunization groups and a mid-level of cellular response. In contrast, the orally DNA vaccine evoked vigorous T cell response and a weak antibody production. These results indicated that the distinct types of immune responses were generated by the different routes of DNA immunization. In addition, our results also show that the delivery of DNA vaccines by electroporation and orally using live-attenuated Salmonella in vivo is an effective method to increase the immune responses. Further studies could be carried out using a combination strategy of both oral and electroporation immunizations to stimulate higher cellular and humoral immune responses.     

Repeated DNA therapeutic vaccination of chronically SIV-infected macaques provides additional virological benefit

We have previously reported that therapeutic immunization by intramuscular injection of optimized plasmid DNAs encoding SIV antigens effectively induces immune responses able to reduce viremia in antiretroviral therapy (ART)-treated SIVmac251-infected Indian rhesus macaques. We subjected such therapeutically immunized macaques to a second round of therapeutic vaccination using a combination of plasmids expressing SIV genes and the IL-15/IL-15 receptor alpha as molecular adjuvant, which were delivered by the more efficacious in vivo constant-current electroporation. A very strong induction of antigen-specific responses to Gag, Env, Nef, and Pol, during ART (1.2–1.6% of SIV-specific T cells in the circulating T lymphocytes) was obtained with the improved vaccination method. Immunological responses were characterized by the production of IFN-γ, IL-2, and TNF-α either alone, or in combination as double or triple cytokine positive multifunctional T cells. A significant induction of CD4⁺ T cell responses, mainly targeting Gag, Nef, and Pol, as well as of CD8⁺ T cells, mainly targeting Env, was found in both T cells with central memory and effector memory markers. After release from ART, the animals showed a virological benefit with a further ∼1 log reduction in viremia. Vaccination with plasmid DNAs has several advantages over other vaccine modalities, including the possibility for repeated administration, and was shown to induce potent, efficacious, and long-lasting recall immune responses. Therefore, these data support the concept of adding DNA vaccination to the HAART regimen to boost the HIV-specific immune responses.     

Protective immunity induced by concurrent intradermal injection of porcine circovirus type 2 and Mycoplasma hyopneumoniae inactivated vaccines in pigs

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.     

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8⁺ T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8⁺ T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines.     

Enhanced immunogenicity and protective efficacy of a tetravalent dengue DNA vaccine using electroporation and intradermal delivery

Phase 1 clinical trials with a DNA vaccine for dengue demonstrated that the vaccine is safe and well tolerated, however it produced less than optimal humoral immune responses. To determine if the immunogenicity of the tetravalent dengue DNA vaccine could be enhanced, we explored alternate, yet to be tested, methods of vaccine administration in non-human primates. Animals were vaccinated on days 0, 28 and 91 with either a low (1 mg) or high (5 mg) dose of vaccine by the intradermal or intramuscular route, using either needle-free injection or electroporation devices. Neutralizing antibody, IFN-γ T cell and memory B cell responses were compared to a high dose group vaccinated with a needle-free intramuscular injection delivery device similar to what had been used in previous preclinical and clinical studies. All previously untested vaccination methodologies elicited improved immune responses compared to the high dose needle-free intramuscular injection delivery group. The highest neutralizing antibody responses were observed in the group that was vaccinated with the high dose formulation via intradermal electroporation. The highest IFN-γ T cell responses were also observed in the high dose intradermal electroporation group and the CD8⁺ T cells were the dominant contributors for the IFNγ response. Memory B cells were detected for all four serotypes. More than a year after vaccination, groups were challenged with dengue-1 virus. Both the low and high dose intradermal electroporation groups had significantly fewer days of dengue-1 virus RNAemia compared to the control group. The results from this study demonstrate that using either an electroporation device and/or the intradermal route of delivery increases the immune response generated by this vaccine in non-human primates and should be explored in humans.     

Retinaldehyde dehydrogenase 2 as a molecular adjuvant for enhancement of mucosal immunity during DNA vaccination

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8⁺ T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8⁺ T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites.     

Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques

Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.     

Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines

Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.     

Partial efficacy of a VSV-SIV/MVA-SIV vaccine regimen against oral SIV challenge in infant macaques

Despite antiretroviral medications, the rate of pediatric HIV-1 infections through breast-milk transmission has been staggering in developing countries. Therefore, the development of a vaccine to protect vulnerable infant populations should be actively pursued. We previously demonstrated that oral immunization of newborn macaques with vesicular stomatitis virus expressing simian immunodeficiency virus genes (VSV-SIV) followed 2 weeks later by an intramuscular boost with modified vaccinia ankara virus expressing SIV (MVA-SIV) successfully induced SIV-specific T and B cell responses in multiple lymphoid tissues, including the tonsil and intestine [13]. In the current study, we tested the oral VSV-SIV prime/systemic MVA-SIV boost vaccine for efficacy against multiple oral SIVmac251 challenges starting two weeks after the booster vaccination. The vaccine did not prevent SIV infection. However, in vaccinated infants, the level of SIV-specific plasma IgA (but not IgG) at the time of challenge was inversely correlated with peak viremia. In addition, the levels of SIV-specific IgA in saliva and plasma were inversely correlated with viral load at euthanasia. Animals with tonsils that contained higher frequencies of SIV-specific TNF-α- or IFN-γ-producing CD8⁺ T cells and central memory T cells at euthanasia also had lower viremia. Interestingly, a marked depletion of CD25⁺FoxP3⁺CD4⁺ T cells was observed in the tonsils as well as the intestine of these animals, implying that T regulatory cells may be a major target of SIV infection in infant macaques. Overall, the data suggest that, in infant macaques orally infected with SIV, the co-induction of local antiviral cytotoxic T cells and T regulatory cells that promote the development of IgA responses may result in better control of viral replication. Thus, future vaccination efforts should be directed towards induction of IgA and mucosal T cell responses to prevent or reduce virus replication in infants.     

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn^®MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p < 0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p < 0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.     

Adjuvant effect of Japanese herbal medicines on the mucosal type 1 immune responses to human papillomavirus (HPV) E7 in mice immunized orally with Lactobacillus-based therapeutic HPV vaccine in a synergistic manner

The Japanese herbal medicines, Juzen-taiho-to (JTT) and Hochu-ekki-to (HET), have been shown to enhance humoral immune responses to vaccine antigen when used as adjuvants for prophylactic vaccines. However, their adjuvant effect on mucosal cellular immune responses remains unstudied. The precursor lesion of cervical cancer, high-grade CIN that expresses HPV E7 oncoprotein ubiquitously is a target for HPV therapeutic vaccines that elicit mucosal E7-specific type 1 T cell responses. We have demonstrated that oral immunization with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7) is more effective in eliciting mucosal E7-specific IFNγ-producing cells than subcutaneous or intramuscular antigen delivery. Here we report the synergistic effect of an oral Lactobacillus-based vaccine and Japanese herbal medicines on mucosal immune responses. Oral immunization of mice with LacE7 plus either a Japanese herbal medicine (JTT or HET) or a mucosal adjuvant, heated-labile enterotoxin T subunit (LTB), promotes systemic E7-specific type 1 T cell responses but not mucosal responses. Administration of LacE7 plus either Japanese herbal medicine and LTB enhanced mucosal E7-specific type 1 T cell response to levels approximately 3-fold higher than those after administration of LacE7 alone. Furthermore, secretion of IFNγ and IL-2 into the intestinal lumen was observed after oral administration of LacE7 and was enhanced considerably by the addition of Japanese herbal medicines and LTB. Our data indicated that Japanese herbal medicines, in synergy with Lactobacillus and LTB, enhance the mucosal type 1 immune responses to orally immunized antigen. Japanese herbal medicines may be excellent adjuvants for oral Lactobacillus-based vaccines and oral immunization of LacE7, HET and LTB may have the potential to elicit extremely high E7-specific mucosal cytotoxic immune response to HPV-associated neoplastic lesions.     

Induction of immune memory by a multisubunit chlamydial vaccine

We tested the hypothesis that intramuscular immunization with a multisubunit chlamydial vaccine candidate will induce long lasting immune responses in mice. Accordingly, groups of female C57BL/6 mice were immunized intramuscularly with Vibrio cholerae ghosts (VCG) expressing the Poring B and polymorphic membrane protein-D proteins of Chlamydia trachomatis or a control antigen. Humoral and cell-mediated immune responses were evaluated following immunization and after live chlamydial infection. Immunization induced an anamnestic response characterized by chlamydial-specific IgG2a and IgA antibodies in sera and vaginal lavage as well as specific genital and splenic T cell responses. The results also revealed that the local mucosal and systemic cellular and humoral immune effectors induced in mice following immunization with the vaccine candidate are long lasting. Vaccinated mice cleared intravaginal challenge with 10⁵ chlamydial inclusion forming units within 12 days compared to control mice, which shed up to 2 × 10³ IFUs at this time point. Moreover, rechallenge of mice 98 days after resolution of the primary infection resulted in the recall and retention of a relatively high frequency of chlamydial-specific Th1 cells and IgG2a in the genital mucosa. These results provide the first evidence that a VCG-based multisubunit chlamydial vaccine is capable of effectively stimulating anamnestic systemic and mucosal immune responses in mice. The data support further vaccine evaluation and testing for induction of long-term protective immunity.     

Mucosal priming with replicative Tiantan vaccinia and systemic boosting with DNA vaccine raised strong mucosal and systemic HIV-specific immune responses

An effective vaccine strategy for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity at mucosal and systemic sites. We tested a new prime-boost approach of intranasal priming with 3 × 10⁶ PFU of replicative recombinant Tiantan vaccinia virus (rTTV) and intramuscular boosting with 100 μg DNA plasmid expressing HIV-1 Gag in BALB/c mice along with other strategies. Our data demonstrated that intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine raised the highest vaginal IgA and systemic T-cell responses, and modest lung IgA and sera IgG responses among all vaccination regimens; each vaccination regimen generated its own imprint of the most preferential T-cell receptor usage of Vβ. These results demonstrate that the combination of intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine is a preferable regimen for induction of both T-cell and humoral immune responses at mucosal and systemic sites.     

Integration of needle-free jet injection with advanced electroporation delivery enhances the magnitude,kinetics, and persistence of engineered DNA vaccine induced immune responses

The combination of optimized DNA constructs, improved formulations and advanced in vivo electroporation (EP) has been shown to generate potent and efficacious immune responses in the clinic. Needle-free jet injection has also been reported to improve DNA vaccine delivery over standard needle and syringe in clinical trials. Here we investigated the impact of combined jet injection and EP (Jet-EP) delivery on muscle transfection efficiency and DNA vaccine immunogenicity in rabbits and nonhuman primates (NHPs) compared to jet injection alone. Our results show that the addition of EP significantly enhanced in vivo DNA transfection efficiency of rabbit muscle over jet injection alone. Jet-EP delivery augmented the rate and magnitude of DNA vaccine induced humoral and cellular responses over jet injection alone in both rabbits and NHPs. Jet-EP delivery also resulted in higher proportions of polyfunctional antigen specific T cells producing IFNγ, IL-2, and/or TNFα. Elevated antibody levels were sustained nine months post immunization in NHPs immunized with a DNA vaccine using Jet-EP delivery, far outperforming jet delivery alone. Our results provide proof-of-concept that addition of advanced EP to needle-free jet injection delivery improves in vivo DNA transfection efficiency, increasing the magnitude, rate and duration of cellular and humoral immune responses to DNA vaccines. This combination likely has significant advantages in important vaccine and immunotherapy settings.     

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1 μg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1 μg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.     

Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: Implication for designing novel mucosal MERS vaccines

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was originally identified in Saudi Arabia in 2012. It has caused MERS outbreaks with high mortality in the Middle East and Europe, raising a serious concern about its pandemic potential. Therefore, development of effective vaccines is crucial for preventing its further spread and future pandemic. Our previous study has shown that subcutaneous (s.c.) vaccination of a recombinant protein containing receptor-binding domain (RBD) of MERS-CoV S fused with Fc of human IgG (RBD-Fc) induced strong systemic neutralizing antibody responses in vaccinated mice. Here, we compared local and systemic immune responses induced by RBD-Fc via intranasal (i.n.) and s.c. immunization pathways. We found that i.n. vaccination of MERS-CoV RBD-Fc induced systemic humoral immune responses comparable to those induced by s.c. vaccination, including neutralizing antibodies, but more robust systemic cellular immune responses and significantly higher local mucosal immune responses in mouse lungs. This study suggests the potential of developing MERS-CoV RBD protein into an effective and safe mucosal candidate vaccine for prevention of respiratory tract infections caused by MERS-CoV.     

人巨细胞病毒IE1核酸疫苗的免疫效果研究

[目的]用人巨细胞病毒(HCMV)IE1核酸疫苗pcDNA3.1(-)-IE1免疫BALB/c小鼠,初步研究其产生的体液免疫和细胞免疫应答水平。[方法]将pcDNA3.1(-)-IE1通过肌肉注射免疫BALB/c小鼠,通过PCR测定和免疫组化检测其在肌细胞中的表达,细胞因子测定、淋巴细胞转化实验检测免疫效果。[结果]PCR检测到与IE1大小一致的片段,免疫组化结果显示IE1基因在小鼠肌细胞中表达IE1目的蛋白。小鼠脾淋巴细胞经PHA刺激后,实验组IL-4、IL-2、IFN-γ含量以及淋巴细胞转化率显著增高,与对照组比较差异有统计学意义(P﹤0.05)。[结论]pcDNA3.1(-)-IE1核酸疫苗能在BALB/c小鼠肌细胞中稳定存在并能表达HCMV IE1蛋白,pcDNA3.1(-)-IE1核酸疫苗可诱导BALB/c小鼠脾细胞分泌IL-4、IL-2、IFN-r并刺激BALB/c小鼠脾细胞增殖。