Characterization of immune responses elicited in mice by intranasal co-immunization with HIV-1 Tat, gp140 ΔV2Env and/or SIV Gag proteins and the nontoxicogenic heat-labile Escherichia coli enterotoxin
Introduction
Since the inexorable spreading of HIV pandemic is unabated, the urgency of designing an effective, safe, inexpensive and easily administrable vaccine to protect people from HIV and/or AIDS is an absolute priority and it has turned out to be an enormous challenge for the scientific community.
The majority of vaccine approaches tested so far aimed at preventing infection by inducing neutralizing antibodies against envelope (Env) antigens (sterilizing immunity). However, their use as cross-clade vaccine candidates is limited due to the high variability of the Env proteins among different HIV clades. Further, the shielding of sensitive epitopes, because of the heavy glycosylation and the very time- and space-constrained exposure of functional domains relevant to neutralization, make even a clade-specific vaccine hard to achieve [1]. As a consequence, the first phase III trial with a monomeric wild-type gp120-based vaccine was a complete failure [2]. HIV-1 Env trimers of gp120 fused with the ectodomain of gp41 are superior to monomeric gp120 for inducing antibody responses directed to conformational epitopes [3], [4] and neutralizing antibodies against R5 and X4 HIV isolates [5], [6]. Nonetheless, cross-clade and broadly neutralizing antibodies are mainly directed against epitopes present in the V3 region of Env [7], [8] and these conserved epitopes are masked and not accessible to neutralization antibodies in native gp120 proteins because of Env conformation and high glycosilation [9], [10].
A substantial advancement in the design of an effective Env-based vaccine comes from studies aimed at generating new immunogens capable of inducing broadly reactive neutralizing antibodies against cryptic neutralization-sensitive epitopes. Indeed, novel variants of trimeric Env from clades B and C were recently generated with a deletion in the V2 region (ΔV2Env) and shown in preclinical models to elicit broader neutralizing antibodies against primary isolates of HIV as compared to their wild-type counterparts [5], [6], [11], [12], [13], [14].
On the other hand, evidence also exists that vaccines based on HIV/SIV structural proteins alone (Env and, to a lesser extent, Gag) in some cases have contained virus replication, and that cellular immunity plays an important role in controlling the acute phase of infection and disease progression even in the absence of neutralizing antibodies [15], [16]. Within this frame, immunization strategies capable of limiting virus replication providing protection from disease progression and reducing virus transmission to healthy individuals have been pursued to fight HIV/AIDS (non-sterilizing immunity). In particular, Tat, Rev and Nef are considered ideal targets for novel vaccine strategies because they are the first HIV proteins expressed early after infection, released in the extracellular environment (Tat and Nef), are more conserved in their immunogenic epitopes among different HIV clades than structural proteins and play key roles in the virus life cycle and in the pathogenesis of the disease. Lastly, evidence exists that humoral and cellular responses directed against Tat, Rev and Nef correlate with the non-progression to AIDS [16], [17]. These vaccine strategies have proven to be safe and immunogenic in mice, in non-human primates and in humans, and effective in protecting from pathogenic challenge in monkeys [16], [17], [18].
In this scenario, consensus is growing on the idea that vaccines combining early and late viral products (combined vaccines) may be superior because they target multiple and sensitive viral proteins [19], [20], [21], [22]. With respect to HIV-1 Tat, preclinical and clinical data indicate that it can be an optimal target for combined vaccine strategies against HIV/AIDS. Tat-based vaccines (both protein and DNA) were shown to be safe and immunogenic in preclinical models and effective in controlling virus replication and blocking disease onset in non-human primates [17], [18], [23], [24], [25], [26]. Individuals with antibodies and cellular responses against Tat have lower incidence and risk of progression to AIDS as compared to anti-Tat negative individuals [27], [28]. The Tat protein is conserved in its immunodominant epitopes and very well recognized by sera from African individuals infected with different virus clades [29] reinforcing the concept that a Tat vaccine may actually be a cross-clade vaccine. Furthermore, preventive and therapeutic phase I trials with a biologically active Tat protein administered by the parenteral route have been completed in Italy and the preliminary data showed that the vaccine is safe and induce humoral and cellular immune responses in most of the vaccinees [30] (Ensoli et al., manuscript in preparation; http://www.hiv1tat-vaccines.info/). In addition, recent evidence also indicates that the biologically active Tat protein displays immunomodulatory activities which can be exploited for the development of combined subunit vaccines [31], [32], [33], [34].
Thus, to assess the feasibility of a vaccine approach based on the combination of early and late HIV proteins, in this study we investigated in Balb/c mice the effect of co-immunization with existing vaccine products, Tat and ΔV2Env soluble proteins (already proven to be safe and immunogenic in phase I clinical trials as single antigens), with or without the SIV Gag antigen.
As HIV enters the human body primarily at mucosal sites [35], the development of vaccination strategies able to promote efficient immune responses at both systemic and mucosal levels may represent effective interventions to combat HIV/AIDS [36], [37]. Further, recent data have demonstrated that, regardless of the port of entry, mucosal tissues and in particular the gut, are heavily targeted during the primary infection [38]. Therefore, induction of protective immune responses at the mucosal level is critical to prevent infection or to control initial damage, thereby halting or delaying progression to disease. The intranasal route of administration is considered a simple method to deliver antigens and offers the possibility for increasing vaccine uptake, inducing immune responses at distal sites and for reducing the costs of the vaccine [39]. However, subunit vaccines are generally poorly immunogenic when administered by the mucosal route unless they are associated with potent mucosal adjuvants [37], [40], [41], [42]. A nontoxigenic mutant of heat-labile enterotoxin (LT) from Escherichia coli, LTK63, has proven to be safe and a potent mucosal adjuvant in animals following intranasal administration [43], [44], [45], [46], [47]. Remarkably, recent phase I clinical trials of a trivalent inactivated influenza vaccine delivered with LTK63 have demonstrated its safety also in humans [48].
For these reasons, in this study antigens were delivered intranasally with LTK63 and local and systemic immune responses were compared to those elicited by immunization with Tat, ΔV2Env and Gag alone.
Section snippets
Proteins, peptides and adjuvant
The 86-aa long Tat protein (HTLVIIIB, BH-10 clone) was expressed in Escherichia coli as described [49] and provided by Diatheva (Fano, Italy). The biological activity of each batch of Tat protein was determined by means of a very sensitive method based on Tat uptake by monocyte-derived dendritic cells [31]. To prevent oxidation that occurs easily because Tat contains seven cysteines, the Tat protein was stored lyophilized at −80 °C and resuspended (2 mg/ml) in degassed sterile phosphate buffered
Intranasal co-immunization with Tat, ΔV2Env and/or Gag results in the induction of antibody responses to the co-administered antigens at both the systemic and mucosal levels
One week after the last boost (day 28), anti-Tat IgG titers were generally higher in all groups immunized with the various antigens associations as compared to the group immunized with Tat alone (Fig. 1A). However, a statistically significant increase was observed only in the group vaccinated with the Tat/Gag formulation as compared to immunization with Tat alone (p = 0.0389). Similarly, anti-Env antibody levels were slightly higher in all groups receiving the various antigen combinations as
Discussion
An effective vaccine against HIV/AIDS of proven safety and efficacy in humans still remains elusive. Since a vaccine is the only realistic way to stop the HIV pandemic, multiple strategies are being pursued by the scientific community to address this enormous challenge. In general vaccines based on viral structural gene products (Env/Gag/Pol) alone have failed to prevent infection by HIV/SIV [16], whereas vaccines based on or including viral regulatory gene products (Tat/Rev/Nef) have shown
Acknowledgements
The research activities described in this publication were funded by the Italian Concerted Action on HIV-AIDS Vaccine Development (ICAV), the EC Commission under the VI Framework Programme of Research and Technological Development (2002–2006) [Project no. LSHP-CT-2003-503240, Mucosal Vaccines for Poverty Related Diseases (“MUVAPRED”), and Project no. LSHP-CT-2004-503487, AIDS Vaccine Integrated Project (“AVIP”)], and the National Institute of Health (NIH NO1-AI-95367 and NO1-AI-05396
References (79)
- et al.
Priming B cell-mediated anti-HIV envelope responses by vaccination allows for the long-term control of infection in macaques exposed to a R5-tropic SHIV
Virology
(2004) - et al.
Development of V2-deleted trimeric envelope vaccine candidates from human immunodeficiency virus type 1 (HIV-1) subtypes B and C
Microbes Infect
(2005) - et al.
Nonstructural HIV proteins as targets for prophylactic or therapeutic vaccines
Curr Opin Biotechnol
(2004) Rational vaccine strategies against AIDS: background and rationale
Microbes Infect
(2005)Criteria for selection of HIV vaccine candidates—general principles
Microbes Infect
(2005)Introduction: rational vaccine strategies against AIDS
Microbes Infect
(2005)- et al.
Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env
Microbes Infect
(2005) - et al.
Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant
Vaccine
(2006) - et al.
Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P)
Vaccine
(2001) - et al.
Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys
Vaccine
(2004)
HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers
J Biol Chem
Porgress towards an AIDS mucosal vaccine: an overview
Tubercolosis
Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes
Vaccine
Antibody responses in volunteers induced by nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin
Vaccine
Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins
Immunol Today
Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants
Vaccine
DNA prime and protein boost immunization with innovative polymeric cationic core-shell nanoparticles elicits broad immune responses and strongly enhance cellular responses of HIV-1 tat DNA vaccination
Vaccine
Candidate HIV-1 gp140ΔV2, Gag and Tat vaccines protect against experimental HIV-1/MuLV challenge
Vaccine
Evaluation of immunogenicity and efficacy of combined DNA and adjuvanted protein vaccination in a human immunodeficiency virus type 1/murine leukemia virus pseudotype challenge model
Vaccine
Intranasal immunization with tetanus toxoid and CNF1 as new mucosal adjuvant protects Balb/c mice against lethal challenge
Vaccine
Enhanced cellular immunity to SIV Gag following co-administration of adenoviruses encoding wild-type or mutant HIV Tat and SIV Gag
Virology
Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs
Vaccine
HIV vaccine design and the neutralizing antibody problem
Nat Immunol
Clinical research. A setback and an advance on the AIDS vaccine front
Science
Humoral response to oligomeric human immunodeficiency virus type 1 envelope protein
J Virol
Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers
J Virol
DNA vaccination with the human immunodeficiency virus type 1 SF162DeltaV2 envelope elicits immune responses that offer partial protection from simian/human immunodeficiency virus infection to CD8(+) T-cell-depleted rhesus macaques
J Virol
Improving on nature: focusing the immune response on the V3 loop
Hum Antibodies
Antibody vs. HIV in a clash of evolutionary titans
Proc Natl Acad Sci U S A
HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites
Nature
The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens
Science
The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region
J Virol
Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate
J Virol
Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region
J Virol
Progress and obstacles in the development of an AIDS vaccine
Nat Rev Immunol
Problems and emerging approaches in HIV/AIDS vaccine development
Expert Opin Emerg Drugs
Recent advances in the development of HIV-1 Tat-based vaccines
Curr HIV Res
Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine
Nat Med
The presence of anti-Tat antibodies is predictive of long-term nonprogression to AIDS or severe immunodeficiency: findings in a cohort of HIV-1 seroconverters
J Infect Dis
Cited by (22)
Nano toolbox in immune modulation and nanovaccines
2022, Trends in BiotechnologyCitation Excerpt :One possible explanation is a difference in the mechanism of immunity that is targeted. Large-sized nanomaterials boost humoral immune responses, whereas smaller NPs promote cell-mediated immune protection [72–74]. Larger NPs have a tendency to preferentially generate type 2 T helper (Th2) cell responses [7,75,76].
Human immunodeficiency virus (HIV) immunopathogenesis and vaccine development: A review
2011, VaccineCitation Excerpt :In mice, the Tat protein was observed to broaden T-cell responses directed to Gag and Env and play the role of an adjuvant [456]. HIV Tat vaccines might therefore show better efficacy when combined with other HIV antigens [457,458]. In view of the lack of success of the development of vaccines that would elicit a protective neutralizing antibody response [254], HIV vaccine development shifted towards vaccines that would induce T-cell responses, especially a CD8+ CTL response.
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
2009, VaccineCitation Excerpt :Finally, Tat is a vaccine relevant antigen and the Tat-vaccine has recently completed preventive and therapeutic phase I clinical testing [23,24] and, based on the results, a phase II-proof of concept trial as therapeutic vaccine is currently being conducted in Italy [http://www.hiv1tat-vaccines.info/]. In addition, due to its immunomodulatory properties [25], Tat (as protein or DNA) will soon be tested in humans in combination with Env and/or other HIV antigens [26–30] within the AIDS Vaccine Integrated Project (AVIP) funded by the European Community [http://avip-eu.org]. The results of the present study demonstrate that two inoculations of the DNA/K2 formulation were sufficient to prime efficiently both arms of the immune system since broad and durable humoral responses with high titers were observed after protein boosting.
Induction of humoral and enhanced cellular immune responses by novel core-shell nanosphere- and microsphere-based vaccine formulations following systemic and mucosal administration
2009, VaccineCitation Excerpt :Finally, in some experiments mice were immunized and sacrificed to collect organs for histological and immunohistochemical examinations. The presence of antigen specific antibodies (IgG) in sera and in mucosal (IgA, IgG) vaginal and lung lavages was searched by Enzyme Linked Immunosorbent Assay (ELISA) on mice samples tested individually, as previously described [42–44]. Splenocytes were purified from spleens squeezed on filters (Cell Strainer, 70 μm, Nylon, Becton Dickinson).