Immunogenicity of fractional-dose of inactivated poliomyelitis vaccine made from Sabin strains delivered by intradermal vaccination in Wistar rats.

Reducing the amount of antigen is an important strategy to resolve the present shortage of IPV supply for global polio eradication. In the study, we compared the immunogenicity of adjuvanted and non-adjuvanted fractional-dose of IPV made from Sabin strains (sIPV) by intradermal (ID) administration versus the full-dose of sIPV by intramuscular (IM) administration in rats by comparing seroconversion rates and geometric mean titers (GMTs) of neutralizing antibodies (NAbs). We found that, after the full 0, 1, 2 months schedule immunizations, the seroconversion rates in all groups reached 100% except non-adjuvanted 1/6 dose group. After 2 immunizations, the seroconversion rates in all the adjuvanted fractional-dose groups and the full-dose group reached 100%. The GMTs of NAbs induced by adjuvanted 1/12 fractional-dose and full-dose of sIPV were similar and dynamics of the antibody responses were consistent. We proves that the Th1/Th2 balance was not changed by the administration route by comparing ratios of the IgG subclass. Our study confirms that ID administration could reduce the required amount of antigens, the adjuvanted fractional-dose resulted in earlier and higher antibody response for all serotypes than that of non-adjuvanted fractional-dose, and the NAbs responses elicited by 1/12 dose was comparable to that by full-dose of sIPV.

Determination of Depth-Dependent Intradermal Immunogenicity of Adjuvanted Inactivated Polio Vaccine Delivered by Microinjections via Hollow Microneedles.

PURPOSE: The aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT). METHODS: A novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 µm, or at 400 µm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization. RESULTS: The applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 µm), -volumes (1-100 µL) and -rates (up to 60 µL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold). CONCLUSION: Intradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Establishment of an Antiplasmodial Vaccine Based on PfRH5-Encoding RNA Replicons Stabilized by Cationic Liposomes.

BACKGROUND: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). METHODS: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. RESULTS: The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. CONCLUSION: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.

Cutaneous CpG adjuvant conditioning to enhance vaccine responses.

Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (TEFF) and central memory (TCM) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced TEFF, TCM, as well as total IgG responses. TEFF and TCM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of TCM 'non-responders' observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome.

Skin Vaccination with Ebola Virus Glycoprotein Using a Polyphosphazene-Based Microneedle Patch Protects Mice against Lethal Challenge.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Ameliorated immunity elicited by intradermal inoculation in individuals vaccinated with inactivated SARS-CoV-2 vaccine.

In clinical trials, antibodies against SARS-CoV-2 were almost eliminated in participants six months after immunization with an inactivated SARS-CoV-2 vaccine. The short duration of antibody persistence is an urgent problem. In this study, the problem was solved by intradermal inoculation with trace antigen. Within 72 h after intradermal inoculation, slight inflammatory reactions, such as redness and swelling, were observed at the inoculation site of the participants. On the 7th, 60th and 180th days after inoculation, the antibodies of the participants were detected, and it was found that the neutralizing antibody and ELISA (IgGs) anti-S antibody levels rapidly increased and were maintained for 6 months. These results indicate that there was a SARS-CoV-2-specific immune response in the participants immunized with an inactivated SARS-CoV-2 vaccine, which could be quickly and massively activated by intradermal trace antigen inoculation to produce an effective clinically protective effect.

Dose-Sparing Intradermal DTaP-sIPV Immunization With a Hollow Microneedle Leads to Superior Immune Responses.

Dose-sparing intradermal (ID) vaccination may induce the same immune responses as intramuscular (IM) vaccination, which can increase vaccine supplies and save costs. In this study, rats were immunized with fractional-dose of Sabin-derived IPV combined with diphtheria-tetanus-acellular pertussis vaccine (DTaP-sIPV) intradermally with hollow microneedle devices called MicronJet600 and the vaccine immunogenicity and efficacy were evaluated and compared with those of full-dose intramuscular immunization. We tested levels of antibodies and the subclass distribution achieved via different immunization routes. Furthermore, gene transcription in the lung and spleen, cytokine levels and protection against Bordetella pertussis (B. pertussis) infection were also examined. The humoral immune effect of DTaP-sIPV delivered with MicronJet600 revealed that this approach had a significant dose-sparing effect and induced more effective protection against B. pertussis infection by causing Th1/Th17 responses. In conclusion, ID immunization of DTaP-sIPV with the MicronJet600 is a better choice than IM immunization, and it has the potential to be a new DTaP-sIPV vaccination strategy.

Therapeutic DNA Vaccine Against HPV16-Associated Cancer.

Human papillomavirus (HPV) is a contagious cause of anogenital and oropharyngeal cancers developing from persistently infected and subsequently transformed basal keratinocytes of mucosal epithelium. DNA-based immunotherapy offers great potential for the treatment of persisting HPV infections and associated cancers. Preclinical testing of therapeutic DNA-based HPV-targeted immunotherapy requires robust animal models which mimic HPV-associated cancer disease in humans. Here we describe a detailed protocol of intradermal delivery of a therapeutic DNA vaccine and a grafting model of neoantigen expressing skin to evaluate vaccine efficacy against HPV16 mediated hyperproliferative epithelium in mice.

Comparative immunogenicity analysis of intradermal versus intramuscular administration of SARS-CoV-2 RBD epitope peptide-based immunogen In vivo.

COVID-19 pandemic has caused severe disruption of global health and devastated the socio-economic conditions all over the world. The disease is caused by SARS-CoV-2 virus that belongs to the family of Coronaviruses which are known to cause a wide spectrum of diseases both in humans and animals. One of the characteristic features of the SARS-CoV-2 virus is the high reproductive rate (R0) that results in high transmissibility of the virus among humans. Vaccines are the best option to prevent and control this disease. Though, the traditional intramuscular (IM) route of vaccine administration is one of the effective methods for induction of antibody response, a needle-free self-administrative intradermal (ID) immunization will be easier for SARS-CoV-2 infection containment, as vaccine administration method will limit human contacts. Here, we have assessed the humoral and cellular responses of a RBD-based peptide immunogen when administered intradermally in BALB/c mice and side-by-side compared with the intramuscular immunization route. The results demonstrate that ID vaccination is well tolerated and triggered a significant magnitude of humoral antibody responses as similar to IM vaccination. Additionally, the ID immunization resulted in higher production of IFN-γ and IL-2 suggesting superior cellular response as compared to IM route. Overall, our data indicates immunization through ID route provides a promising alternative approach for the development of self-administrative SARS-CoV-2 vaccine candidates.

Intradermal Immunization of EBOV VLPs in Guinea Pigs Induces Broader Antibody Responses Against GP Than Intramuscular Injection.

Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection.

Diphtheria toxoid dissolving microneedle vaccination: Adjuvant screening and effect of repeated-fractional dose administration.

In this study the effect of repeated-fractional intradermal administration of diphtheria toxoid (DT) compared to a single administration in the presence or absence of adjuvants formulated in dissolving microneedles (dMNs) was investigated. Based on an adjuvant screening with a hollow microneedle (hMN) system, poly(I:C) and gibbsite, a nanoparticulate aluminum salt, were selected for further studies: they were co-encapsulated with DT in dMNs with either a full or fractional DT-adjuvant dose. Sharp dMNs were prepared regardless the composition and were capable to penetrate the skin, dissolve within 20 min and deposit the intended antigen-adjuvant dose, which remained in the skin for at least 5 h. Dermal immunization with hMN in repeated-fractional dosing (RFrD) resulted in a higher immune response than a single-full dose (SFD) administration. Vaccination by dMNs led overall to higher responses than hMN but did not show an enhanced response after RFrD compared to a SFD administration. Co-encapsulation of the adjuvant in dMNs did not increase the immune response further. Immunization by dMNs without adjuvant gave a comparable response to subcutaneously injected DT-AlPO4 in a 15 times higher dose of DT, as well as subcutaneous injected DT-poly(I:C) in a similar DT dose. Summarizing, adjuvant-free dMNs showed to be a promising delivery tool for vaccination performed in SFD administration.

A DNA Vaccine Encoding Plasmodium falciparum PfRH5 in Cationic Liposomes for Dermal Tattooing Immunization.

Vaccines are the primary means of controlling and preventing pandemics and outbreaks of pathogens such as bacteria, viruses, and parasites. However, a major drawback of naked DNA-based vaccines is their low immunogenicity and the amount of plasmid DNA necessary to elicit a response. Nano-sized liposomes can overcome this limitation, enhancing both nucleic acid stability and targeting to cells after administration. We tested two different DNA vaccines in cationic liposomes to improve the immunogenic properties. For this, we cloned the coding sequences of the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) either alone or fused with small the small hepatitis virus (HBV) envelope antigen (HBsAg) encoding sequences, potentially resulting in HBsAg particles displaying PfRH5 on their outside. Instead of invasive intraperitoneal or intramuscular immunization, we employed intradermal immunization by tattooing nano-encapsulated DNA. Mice were immunized with 10 µg encapsulated DNA encoding PfRH5 alone or in fusion with HBsAg and this elicited antibodies against schizont extracts (titer of 104). Importantly, only IgG from animals immunized with PfRH5-HBs demonstrated sustained IgG-mediated inhibition in in vitro growth assays showing 58% and 39% blocking activity after 24 and 48 h, respectively. Intradermal tattoo-vaccination of encapsulated PfRH5-HBsAg coding plasmid DNA is effective and superior compared with an unfused PfRH5-DNA vaccine, suggesting that the HBsAg fusion may be advantageous with other vaccine antigens.

Intradermal active full-length DNA Aß42 immunization via electroporation leads to high anti-Aß antibody levels in wild-type mice.

Aß immunotherapies with anti-Aß antibody responses have high potential as possible prevention treatment for Alzheimer's disease. We have previously shown that active DNA Aß1-42 immunization via gene gun delivery led to a non-inflammatory immune response resulting in decreased Aß levels in brains of an immunized AD mouse model. To make DNA vaccination more applicable for clinical use, we used here intradermal electroporation. With fine tuning of the electropulse parameters, high antibody levels and low levels of inflammatory cytokines in the cellular immunoassays were observed. Full-length DNA Aß1-42 immunization delivered via electroporation has potential to be used in the clinical setting.

Hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded PLGA nanoparticles elicits protective T cell-mediated immunity to an intracellular bacterium.

The skin is an attractive organ for immunization due to the presence of a large number of epidermal and dermal antigen-presenting cells. Hollow microneedles allow for precise and non-invasive intradermal delivery of vaccines. In this study, ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with and without TLR3 agonist poly(I:C) were prepared and administered intradermally by hollow microneedles. The capacity of the PLGA nanoparticles to induce a cytotoxic T cell response, contributing to protection against intracellular pathogens, was examined. We show that a single injection of OVA-loaded PLGA nanoparticles, compared to soluble OVA, primed both adoptively transferred antigen-specific naïve transgenic CD8+ and CD4+ T cells with markedly high efficiency. Applying a triple immunization protocol, PLGA nanoparticles primed also endogenous OVA-specific CD8+ T cells. Immune response, following immunization with in particular anionic PLGA nanoparticles co-encapsulated with OVA and poly(I:C), provided protection against a recombinant strain of the intracellular bacterium Listeria monocytogenes, secreting OVA. Taken together, we show that PLGA nanoparticle formulation is an excellent delivery system for protein antigen into the skin and that protective cellular immune responses can be induced using hollow microneedles for intradermal immunizations.

Diphtheria toxoid and N-trimethyl chitosan layer-by-layer coated pH-sensitive microneedles induce potent immune responses upon dermal vaccination in mice.

Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH5.8 and nearly uncharged at pH7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ±0.6µg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5µg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization.

Repeated fractional intradermal dosing of an inactivated polio vaccine by a single hollow microneedle leads to superior immune responses.

The purpose of this study was to investigate the effect of various repeated fractional intradermal dosing schedules of inactivated polio vaccine serotype 1 (IPV1) on IPV1-specific IgG responses in rats. By utilizing an applicator that allowed for precisely controlled intradermal microinjections by using a single hollow microneedle, rats were immunized intradermally with 5 D-antigen units (DU) of IPV1 at 150µm skin depth. This dose was administered as a bolus, or in a repeated fractional dosing schedule: 4 doses of 1.25 DU (1/4th of total dose) were administered on four consecutive days or every other day; 8 doses of 0.625 DU (1/8th of total dose) were administered on eight consecutive days; or 4 exponentially increasing doses (0.04, 0.16, 0.8 and 4 DU), either with or without an exponentially increasing CpG oligodeoxynucleotide 1826 (CpG) dose, were administered on four consecutive days. All of these fractional dosing schedules resulted in up to ca. 10-fold higher IPV1-specific IgG responses than intradermal and intramuscular bolus dosing. IPV1 combined with adjuvant CpG in exponential dosing did not significantly increase the IPV1-specific IgG responses further, which demonstrated that maximal responses were achieved by fractional dosing. In conclusion, repeated fractional intradermal IPV1 dosing leads to superior IPV1-specific IgG responses without the use of adjuvants. These results indicate that a controlled release delivery system for intradermal IPV1 delivery can potentiate IPV1-specific IgG responses.

Multifunctional particle-constituted microneedle arrays as cutaneous or mucosal vaccine adjuvant-delivery systems.

To overcome drawbacks of current injection vaccines, such as causing needle phobia, needing health professionals for inoculation, and generating dangerous sharps wastes, researchers have designed novel vaccines that are combined with various microneedle arrays (MAs), in particular, with the multifunctional particle-constructed MAs (MPMAs). MPMAs prove able to enhance vaccine stability through incorporating vaccine ingredients in the carrier, and can be painlessly inoculated by minimally trained workers or by self-administration, leaving behind no metal needle pollution while eliciting robust systemic and mucosal immunity to antigens, thanks to delivering vaccines to cutaneous or mucosal compartments enriched in professional antigen-presenting cells (APCs). Especially, MPMAs can be easily integrated with functional molecules fulfilling targeting vaccine delivery or controlling immune response toward a Th1 or Th2 pathway to generate desired immunity against pathogens. Herein, we introduce the latest research and development of various MPMAs which are a novel but promising vaccine adjuvant delivery system (VADS).

Skin Immunization Obviates Alcohol-Related Immune Dysfunction.

Alcoholics suffer from immune dysfunction that can impede vaccine efficacy. If ethanol (EtOH)-induced immune impairment is in part a result of direct exposure of immune cells to EtOH, then reduced levels of exposure could result in less immune dysfunction. As alcohol ingestion results in lower alcohol levels in skin than blood, we hypothesized that the skin immune network may be relatively preserved, enabling skin-targeted immunizations to obviate the immune inhibitory effects of alcohol consumption on conventional vaccines. We employed the two most common chronic EtOH mouse feeding models, the liver-damaging Lieber-DeCarli (LD) and liver-sparing Meadows-Cook (MC) diets, to examine the roles of EtOH and/or EtOH-induced liver dysfunction on alcohol related immunosuppression. Pair-fed mice were immunized against the model antigen ovalbumin (OVA) by DNA immunization or against flu by administering the protein-based influenza vaccine either systemically (IV, IM), directly to liver (hydrodynamic), or cutaneously (biolistic, ID). We measured resulting tissue EtOH levels, liver stress, regulatory T cell (Treg), and myeloid-derived suppressor cell (MDSC) populations. We compared immune responsiveness by measuring delayed-type hypersensitivity (DTH), antigen-specific cytotoxic T lymphocyte (CTL), and antibody induction as a function of delivery route and feeding model. We found that, as expected, and independent of the feeding model, EtOH ingestion inhibits DTH, CTL lysis, and antigen-specific total IgG induced by traditional systemic vaccines. On the other hand, skin-targeted vaccines were equally immunogenic in alcohol-exposed and non-exposed subjects, suggesting that cutaneous immunization may result in more efficacious vaccination in alcohol-ingesting subjects.

Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant.

Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.