Potent Cellular Immune Responses Research Articles

Vaccines expressing the innate immune modulator EAT-2 elicit potent effector memory T lymphocyte responses despite pre-existing vaccine immunity.

The mixed results from recent vaccine clinical trials targeting HIV-1 justify the need to enhance the potency of HIV-1 vaccine platforms in general. Use of first-generation recombinant adenovirus serotype 5 (rAd5) platforms failed to protect vaccinees from HIV-1 infection. One hypothesis is that the rAd5-based vaccine failed due to the presence of pre-existing Ad5 immunity in many vaccines. We recently confirmed that EAT-2-expressing rAd5 vectors uniquely activate the innate immune system and improve cellular immune responses against rAd5-expressed Ags, inclusive of HIV/Gag. In this study, we report that use of the rAd5-EAT-2 vaccine can also induce potent cellular immune responses to HIV-1 Ags despite the presence of Ad5-specific immunity. Compared to controls expressing a mutant SH2 domain form of EAT-2, Ad5 immune mice vaccinated with an rAd5-wild-type EAT-2 HIV/Gag-specific vaccine formulation significantly facilitated the induction of several arms of the innate immune system. These responses positively correlated with an improved ability of the vaccine to induce stronger effector memory T cell-biased, cellular immune responses to a coexpressed Ag despite pre-existing anti-Ad5 immunity. Moreover, inclusion of EAT-2 in the vaccine mixture improves the generation of polyfunctional cytolytic CD8(+) T cell responses as characterized by enhanced production of IFN-γ, TNF-α, cytotoxic degranulation, and increased in vivo cytolytic activity. These data suggest a new approach whereby inclusion of EAT-2 expression in stringent human vaccination applications can provide a more effective vaccine against HIV-1 specifically in Ad5 immune subjects.

Mathematical Models of E-Antigen Mediated Immune Tolerance and Activation following Prenatal HBV Infection

We develop mathematical models for the role of hepatitis B e-antigen in creating immunological tolerance during hepatitis B virus infection and propose mechanisms for hepatitis B e-antigen clearance, subsequent emergence of a potent cellular immune response, and the effect of these on liver damage. We investigate the dynamics of virus-immune cells interactions, and derive parameter regimes that allow for viral persistence. We modify the model to account for mechanisms responsible for hepatitis B e-antigen loss, such as seroconversion and virus mutations that lead to emergence of cellular immune response to the mutant virus. Our models demonstrate that either seroconversion or mutations can induce immune activation and that instantaneous loss of e-antigen by either mechanism is associated with least liver damage and is therefore more beneficial for disease outcomes.

Accelerated Heterologous Adenovirus Prime-Boost SIV Vaccine in Neonatal Rhesus Monkeys

A pediatric human immunodeficiency virus type 1 (HIV-1) vaccine would be desirable to protect infants against HIV-1 transmission from breast-feeding. Such a vaccine would need to induce protective immunity at mucosal surfaces in neonates as soon as possible after birth. Recombinant adenovirus (rAd) vectors have been shown to elicit potent systemic and mucosal virus-specific immune responses in adult nonhuman primates and humans, but these vectors have not previously been comprehensively studied in infants. In this study, we demonstrate that a single injection of rAd26 encoding simian immunodeficiency virus mac239 (SIVmac239) Gag on the day of birth elicited detectable Gag-specific cellular immune responses in rhesus monkeys, but these responses were transient and waned quickly. In contrast, an accelerated heterologous prime-boost regimen involving administration of rAd35 at birth and rAd26 at 4 weeks of life elicited potent and durable Gag-specific cellular and humoral immune responses in neonatal rhesus monkeys, including mucosal responses that remained detectable at 1 year of age. These results suggest the potential of an accelerated heterologous rAd prime-boost regimen as a candidate HIV-1 vaccine for newborns.

Nanoparticle vaccines enhance humoral responses to a malaria antigen by expanding Tfh cells and inducing germinal center formation (166.10)

Abstract Nanoparticles (NP) are promising delivery vehicles for vaccines that promote humoral and cellular immunity in concert. We recently developed a novel lipid-based NP vaccine system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs). ICMVs efficiently entrap and co-deliver antigens and molecular adjuvants to lymphoid tissues, initiating potent cellular and humoral immune responses. ICMVs were synthesized by fusing liposomes containing antigen into multilamellar vesicles, followed by bilayer-to-bilayer crosslinking of lipid bilayers within the vesicle walls to stabilize the particles. ICMVs carrying ovalbumin and the TLR agonist MPLA induced robust cytotoxic T lymphocyte responses in mice, achieving a peak ~30% tetramer+ cells in the CD8+ T-cell population after 3 immunizations. Mice vaccinated with ICMVs containing a candidate Plasmodium vivax antigen raised high-titer, high-avidity antibody responses against the antigen lasting > 1 yr, at 10-fold lower dose of antigen than conventional adjuvants such as alum or Montanide. Compared to vaccination with alum, immunization with ICMVs prolonged antigen accumulation at the draining lymph nodes, enhanced germinal center formation, and increased the expansion of antigen-specific follicular helper T-cells, partially explaining the enhanced antibody response. These results suggest that antigen/adjuvant-carrying ICMVs may serve as a platform to elicit potent cellular and humoral immune responses against infectious pathogens.

Gene Therapy Research at the Frontiers of Viral Immunology

Gene Therapy Research at the Frontiers of Viral Immunology

Study on vector-specific and foreign gene-specific immune responses induced by rAd5 and rAAV2/1 vaccines

To compare the foreign gene-specific and vector-specific immune responses in BALB/c mice immunized with rAd5 or rAAV2/1 expressing the same gene. BALB/c mice were immunized with rAd5-gag or rAAV2/1-gag once, HIV-1 Gag-specific and vector-specific cellular immune responses were analyzed by Elispot assay, HIV-1 P24-specific IgG and vector-specific IgG were tested by ELISA assay. Mice immunized with rAd5-gag induced potent Gag-specific cellular immune responses and that were significantly higher than Ad5-specfic cellular responses, while rAAV2/1-gag elicited weak Gag-specific and AAV2/1-specific cellular responses. Both P24-specific and Ad5-specific IgG titers induced by rAd5-gag were high and in similar level. Higher level of P24-specific IgG was found in mice inoculated with rAAV2/1-gag than rAd5-gag. And the P24-specific IgG titers were higher than the vector-specific IgG titers in mice immunized with rAAV2/1. rAd5 could elicit strong foreign gene-specific cellular and humoral immune responses, weak vector-specific cellular responses and strong vector-specific antibodies, rAAV2/1 could induce potent foreign gene-specific antibodies that were much higher than vector-specific IgG, while both foreign gene-specific and vector-specific cellular responses were very low.

Intranasal Sendai viral vector vaccination is more immunogenic than intramuscular under pre-existing anti-vector antibodies

Viral vectors are promising vaccine tools for eliciting potent cellular immune responses. Pre-existing anti-vector antibodies, however, can be an obstacle to their clinical use in humans. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient CD8(+) T-cell induction in macaques. Here, we investigated the immunogenicity of SeV vector vaccination in the presence of anti-SeV antibodies. We compared antigen-specific CD8(+) T-cell responses after intranasal or intramuscular immunization with a lower dose (one-tenth of that in our previous studies) of SeV vector expressing simian immunodeficiency virus Gag antigen (SeV-Gag) between naive and pre-SeV-infected cynomolgus macaques. Intranasal SeV-Gag immunization efficiently elicited Gag-specific CD8(+) T-cell responses not only in naive but also in pre-SeV-infected animals. In contrast, intramuscular SeV-Gag immunization induced Gag-specific CD8(+) T-cell responses efficiently in naive but not in pre-SeV-infected animals. These results indicate that both intranasal and intramuscular SeV administrations are equivalently immunogenic in the absence of anti-SeV antibodies, whereas intranasal SeV vaccination is more immunogenic than intramuscular in the presence of anti-SeV antibodies. It is inferred from a recent report investigating the prevalence of anti-SeV antibodies in humans that SeV-specific neutralizing titers in more than 70% of people are no more than those at the SeV-Gag vaccination in pre-SeV-infected macaques in the present study. Taken together, this study implies the potential of intranasal SeV vector vaccination to induce CD8(+) T-cell responses even in humans, suggesting a rationale for proceeding to a vaccine clinical trial using this vector.

Identification and Immunological Characterization of Three Potential Vaccinogens against Cryptosporidium Species

Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and Cryptosporidium parvum, leading to acute, persistent, and chronic diarrhea with life-threatening consequences in immunocompromised individuals. In developing countries, cryptosporidiosis in early childhood has been associated with subsequent significant impairment in growth, physical fitness, and intellectual abilities. Currently, vaccines are unavailable and chemotherapeutics are toxic and impractical, and agents for immunoprophylaxis or treatment of cryptosporidiosis are a high priority. Availability of the genome sequences for C. hominis and C. parvum provides new opportunities to procure and examine novel vaccine candidates. Using the novel approach of "reverse vaccinology," we identified several new potential vaccine candidates. Three of these antigens--Cp15, profilin, and a Cryptosporidium apyrase--were delivered in heterologous prime-boost regimens as fusions with cytolysin A (ClyA) in a Salmonella live vaccine vector and as purified recombinant antigens, and they were found to induce specific and potent humoral and cellular immune responses, suggesting their potential as new vaccinogens against Cryptosporidium infection.

Durable Mucosal Simian Immunodeficiency Virus-Specific Effector Memory T Lymphocyte Responses Elicited by Recombinant Adenovirus Vectors in Rhesus Monkeys

The induction of potent and durable cellular immune responses in both peripheral and mucosal tissues may be important for the development of effective vaccines against human immunodeficiency virus type 1 and other pathogens. In particular, effector responses at mucosal surfaces may be critical to respond rapidly to incoming mucosal pathogens. Here we report that intramuscular injection of nonreplicating recombinant adenovirus (rAd) vectors into rhesus monkeys induced remarkably durable simian immunodeficiency virus (SIV)-specific T lymphocyte responses that persisted for over 2 years in both peripheral blood and multiple mucosal tissues, including colorectal, duodenal, and vaginal biopsy specimens, as well as bronchoalveolar lavage fluid. In peripheral blood, SIV-specific T lymphocytes underwent the expected phenotypic evolution from effector memory T cells (T(EM)) to central memory T cells (TCM) following vaccination. In contrast, mucosal SIV-specific T lymphocytes exhibited a persistent and durable T(EM) phenotype that did not evolve over time. These data demonstrate that nonreplicating rAd vectors induce durable and widely distributed effector memory mucosal T lymphocyte responses that are phenotypically distinct from peripheral T lymphocyte responses. Vaccine-elicited T(EM) responses at mucosal surfaces may prove critical for affording protection against invading pathogens at the mucosal portals of entry.

CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

Vaccines based on recombinant proteins avoid toxicity and anti-vector immunity associated with live vaccine (e.g., viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell (CD8T) responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8T responses comparable to live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles (ICMVs) formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. ICMVs stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying ICMVs form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for protein therapeutic delivery.

Toll Like Receptors Play a Role in General Immunity, Eye Infection and Inflammation: Tlrs for Nanodelivery

Dendritic cells [DCs] are potent antigen presenting cells [APC], which plays a vital role in immune system by detecting and capturing pathogens in the body. DCs perform a pivotal role in induction of T cell response. Regulation of immune response can be achieved by specific antigen [Ag] delivery to DCs. A delivery system that can efficiently target and present Ags to DCs for the purpose of anti-tumour activity is currently a topic of significant research interest. DCs are receiving attention due to their key role in anti cancer host response and due to their adjuvanic property in tumour vaccines. Role of toll like receptors [TLR] in innate immune system and their part in eventual stimulation of adaptive immunity is exploited to develop vaccines. TLR agonists in conjugation with vaccines are shown to increase therapeutic efficacy in some cases. TLRs also play a vital role in protecting the cornea from invading pathogens. Due to adverse effects in the treatment of ocular inflammations, cancer and in viral infections, an alternate approach such as the use of TLRs will solve the inquisitive question regarding side effects. The intended delivery is attained by the use of nanoparticles which in turn leads to prolonged half-life in the body. Co-delivery of Ags, TLRs and immunomodulators using nanoparticles has been demonstrated to elicit potent cellular immune responses and are currently under development of clinically applicable immunisations and vaccines.

Vaccine protection against lethal homologous and heterologous challenge using recombinant AAV vectors expressing codon-optimized genes from pandemic swine origin influenza virus (SOIV)

The recent H1N1 influenza pandemic and the inevitable delay between identification of the virus and production of the specific vaccine have highlighted the urgent need for new generation influenza vaccines that can preemptively induce broad immunity to different strains of the virus. In this study we have produced AAV-based vectors expressing the A/Mexico/4603/2009 (H1N1) hemagglutinin (HA), nucleocapsid (NP) and the matrix protein M1 and have evaluated their ability to induce specific immune response and protect mice against homologous and heterologous challenge. Each of the vaccine vectors elicited potent cellular and humoral immune responses in mice. Although immunization with AAV-M1 did not improve survival after challenge with the homologous strain, immunization with the AAV-H1 and AAV-NP vectors resulted in survival of all mice, as did inoculation with a combination of all three vectors. Furthermore, trivalent vaccination also conferred partial protection against challenge with the highly heterologous and virulent A/PR/8/34 strain of H1N1 influenza.

Robust Vaccine-Elicited Cellular Immune Responses in Breast Milk following Systemic Simian Immunodeficiency Virus DNA Prime and Live Virus Vector Boost Vaccination of Lactating Rhesus Monkeys

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Novel adenovirus vector-based vaccines for HIV-1.

Recombinant adenovirus (rAd) vectors have emerged as promising vaccine platform technologies due to their capacity to elicit potent humoral and cellular immune responses to encoded antigens. These vectors are being explored as potential vaccine candidates for a variety of pathogens. This review summarizes current efforts to develop rAd vector-based vaccines for HIV-1. In the phase 2b Step study, rAd5 vectors expressing clade B HIV-1 Gag, Pol, and Nef antigens failed to afford protection and may have resulted in increased HIV-1 acquisition in certain subgroups. Recent studies have explored the potential reasons for this failure and the utility of novel rAd vectors derived from non-Ad5 serotypes. Current areas of active investigation include the development of alternative serotype rAd vectors, the incorporation of rAd vectors into heterologous vector prime-boost regimens, and the use of rAd vectors to express novel HIV-1 antigens. These HIV-1 vaccine candidates will be evaluated in clinical trials over the next several years.

Immunization with pre-erythrocytic antigen CelTOS from Plasmodium falciparum elicits cross-species protection against heterologous challenge with Plasmodium berghei.

BackgroundThe Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium.Methodology/Principal FindingsTo test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses.Conclusions/SignificanceImmunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species.

New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

Efficient generation of a monoclonal antibody against the human C-type lectin receptor DCIR by targeting murine dendritic cells

Dendritic cells (DCs) are very important for the generation of long lasting immune responses against pathogens or the induction of anti-tumor responses. Targeting antigen to dendritic cells via monoclonal antibodies specific for DC cell surface receptors such as DEC205 was shown to elicit potent cellular and humoral immune responses in vivo. Therefore, we investigated whether this novel strategy might also be useful for the generation of new monoclonal antibodies against molecules of choice. We show, that by targeting the extracellular domain of the human C-type lectin receptor ClecSF6/DCIR/LLIR (hDCIR) to DEC205 on DCs in vivo, we were able to generate highly specific monoclonal antibodies against hDCIR.

Construction of, and T-helper (Th)1/Th2 immune responses to, a herpes simplex virus type 2 glycoprotein D-cytotoxic T-lymphocyte epitope DNA vaccine

Recombinant cytotoxic T lymphocyte (CTL) epitope DNA vaccines offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. To construct a pVAX-gD-CTL vector expressing the glycoprotein (g)D and gB CTL epitopes from herpes simplex virus type 2 (HSV2) and evaluate it in mice for immunogenicity and protective efficacy against intraperitoneal challenge with the HSV2 strain Sav. The gD gene transcript and gB CTL epitope were inserted into the pVAX vector to obtain the recombinant plasmid pVAX-gD-CTL. An in vitro study was then conducted to detect the expression of gD by immunocytochemistry and western blotting. BALB/c mice were immunized with this DNA vaccine, then the CTL activity and expression of anti-HSV2 gD IgG, interferon-gamma and interleukin-4 by lactate dehydrogenase release assay and ELISA, respectively. The protection given to the mice was assayed by a fatal dose of virus. The pVAX-gD-CTL vector was successfully constructed and could express gD in COS-7 cells. Immunogenicity of gD, anti-HSV2 gD neutralizing serum IgG antibody and robust Th1-polarized immune responses were found. Furthermore, mice were prophylactically protected from challenge with a high dose of HSV2. In summary, pVAX-gD-CTL vector was successfully used to elicit potent Th1-like cellular and humoral immune responses that were protective against HSV2 disease in mice.

P16-36. Functional genomic immune profile of SIV DNA vaccines delivered by electroporation and combined with molecular adjuvants

Background There are a number of challenges in the development of an effective vaccine for HIV, including stimulation of potent and broad cellular and humoral immune response that can cross protect between strains or clades. We have studied the cellular immune response, using genomics, of a DNA based vaccine that resulted in potent and broad cellular immune responses that suppressed SIV251 replication.