Molecular characterization and functional analysis of the PilQ380-705: a novel secretin domain in Pseudomonas aeruginosa

Background: Type 4 pili [T4P] is an important virulence factor of Pseudomonas aeruginosa [P. aeruginosa]. T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ [PilQ380-706] was produced as a recombinant protein

Methods: A 981 bp fragment of C-terminal of the pilQ secretin [pilQ1138-2118] from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ1138-2118 gene in the recombinant construct [pET28a/pilQ] was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ380-706 confirmed by Western blot analysis and twitching inhibition assay

Results: The PCR and enzymatic digestion results showed the presence of the pilQ1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ380-706 is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ380-706 protein. The PilQ380-706 protein showed high biological activity in all of these standard assays

Conclusion: Since, the PilQ380-706 protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies

Th1 immune response induction by biogenic selenium nanoparticles in mice with breast cancer: preliminary vaccine model

Background: tumor associated antigens can be viably used to enhance host immune response

Objectives: the immunomodulatory effect of biogenic selenium nanoparticles [SeNPs] was compared between treated and untreated mice with crude antigens of 4T1 cells

Materials and Methods: female inbred BALB/c mice [60] were injected by cancinogenic 4T1 cells causing breast cancer. After 10 days, all tumor bearing mice were divided into 4 groups. Group 1 was daily provided oral PBS and injected by the same buffer after tumor induction and was considered as control. Group 2 received only 100 [micro]g/day SeNPs as an oral supplement for 30 days. Group 3 was only injected with 4T1 cells crude antigens with nil supplementation of SeNPs. Group 4 animals were supplemented 100 [micro]g/day SeNPs for 30 days and simultaneously injected with crude antigens of 4T1 cells. All antigens or PBS injections were introduced at 7, 14 and 28 days following tumor induction. Oral PBS and SeNPs supplementation initiated from the first day of tumor induction and continued up to 30 days. During tumor growth, animal weights and survival rates were monitored and at the end of the study the concentrations of different cytokines and DTH responses were measured

Results: data clearly showed that the levels of cellular immunomodulatory components [granzyme B, IL-12, IFN-[lambada], and IL-2] significantly increased [P < 0.05] in mice treated with both SeNPs and crude antigens of 4T1 cells in comparison to the other groups. In contrast, the levels of TGF-[beta] in these mice decreased

Conclusions: although SeNPs showed a noticeable boosting effect for the immune response in mice bearing tumor exposed to crude antigens of 4T1 cells, further complementary studies seem to be inevitable

Induction of IFN-gamma cytokine response against hepatitis B surface antigen using melittin

In this study we co-administered melittin along with HBsAg/alum vaccine to investigate if it helps elicitation of Th1/Th2 response. Hepatitis B virus [HBV] infection is a life-threatening liver infection, which can lead to chronic liver disease. Vigorous T cell responses are stimulated at acute, self-limiting HBV infection, while chronic HBV infection elicits very weak T cell responses. The prevalence of HBV infection has been decreased by the approved vaccination approach using recombinant HBs antigen [HBsAg] and alum i.e. HBV vaccine. Alum, a strong Th2 stimulator, is usually used as adjuvant to increase HBsAg immunogenicity. The present vaccine does not induce protective and/or prophylactic immune response in some groups. Melittin, major active component in the venom of honeybee, induces Th1 development. Experimental mice were immunized with melittin plus hepatitis B vaccine on day 0 following by two booster doses with the same injections. Lymphocyte proliferation, IFN-gamma, and IL-4 level, total antibody and isotyping of IgG1, IgG2a IgG2b, and IgM were measured using ELISA. Administration of melittin and HBV vaccine had no effect on lymphoproliferation and total antibody responses, but increased IFN-gamma response and induced Th1 response. The present study proposed that administration of melittin along with conventional vaccine shifts T cell responses towards Th1/Th2 dominated with Th1 response. The resultant immune response leads to activation of both cell-mediated and humoral immune responses, both of which required for clearance of HBV infection.

[Efficacy of AJc-F nano-complex in the improvement of hepatitis B vaccine immunogenicity]

Prevalence of hepatitis B infection, existence of non-responder populations and the weakness of the classic Alum adjuvant in inducing proper cellular immune responses display the importance of designing more efficient vaccines. Many studies have demonstrated interactions between iron and the immune system. In the present study, we have evaluated the effects of two iron containing structures, ferrous sulphate and AJc-F nano complex on immune response. In this experimental study, female Balb/c mice were injected 3 times with hepatitis B vaccine. The proliferative response of the splenocytes was evaluated using bromodeoxyuridine assay and interleukin-4, interferon-gamma [IFN-gamma] levels and total specific antibodies were examined by the ELISA method. Data analysis was done by SPSS software version 18. AJc-F nano complex significantly increased total antibody and cytokine levels compared to ferrous sulfate and control groups. Ferrous sulfate severely decreased total antibody and IL-4 levels compared to control group. Both compounds increased splenocyte proliferation and IFN-gamma levels compared to control group. The results show that AJc-F, as an iron-containing nano complex, could efficiently increase humoral and cellular immune responses against hepatitis B vaccine

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

Cloning, expression and purification of penicillin binding proteinla [PBP2A] from methicillin resistant staphylococcus aureus: a study on immunoreactivity in BALB/C mouse

Staphylococcus aureus [S. aureus] is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of 5. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24alpha-mec cplasmid was transformed into competent E.coll BL2l [DE3] cells. Recombinant protein was over expressed with 1 mM isopropythio-beta-D-galctoside [IPTG] and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 pg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies

[Effect of oral administration of Lactobacillus reuteri in increased survival and resistance to neoplasm in mice breast cancer]

Background: Several reports indicate that the probiotics can increase body resistance against malignant tumors. The aim of this study is to evaluate the effect of Lactobaci-llus reuteri Persian type culture collection [PTCC] 1655 in preventing tumor growth, improving weight and survival rate in mice with breast cancer

Methods: Twenty mice, the BALB/c at six weeks age, weighing approximately 17 gram were divided into two groups. Oral administration of 500 micro liters of Lactobacillus reuteri suspension performed for the first group 14 days before tumor transplantation. The second group [control] received the same volume of phosphate buffer saline [PBS]. Then the mice had tumor transplantation surgery. Lactobacillus reuteri was prescribed in the first group in seven-day period and three-day interruption pattern. At the same time the second group [control] received PBS. This process was continued until 45 day. The tumor growth, histology and body weight were evaluated in both group and the mortality of mice was recorded

Results: In the mice transplanted tumors that had received probiotics, tumor growth decreased in comparison with control group. In this group the body weight increased [P>0/05]. In addition, the survival of these mice had significantly increased compared to control group [P=0.002]. The evaluation of tumor tissue also showed increased immune system function in mice receiving the probiotic [P>0/05]

Conclusion: Lactobacillus reuteri can improve immune system function and have an important role to help treatment of cancer

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice

Noradrenaline [NA], the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, antiangiogenic and cytotoxic properties of propranolol, beta-AR blocker, against various cancers. To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxic activity of the splenocytes, naturally occurring CD4+ CD25[high] T-reg cells and IFN-gamma and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. A significant increase in the level of IFN- gamma in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-gamma. Likewise, a decrease in tumor growth in the test group [p<0.01] and a significant increase in CTL activity [p<0.05] was observed. HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses

Heat shock proteins enriched-promastigotes of leishmania major inducing Th2 immune response in BALB/c mice

Heat shock proteins [HSP] are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen [SLA] was determined. BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10[6] stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks. No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-gamma, IL-4, TGF-beta, IgG1 and IgG2b were increased in both groups, IFN-gamma was significantly higher in SLA group and IgG2a in HSP-enriched SLA. These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania major and Expression in the Chinese Hamster Ovary Cell Line

Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting. Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Cloning, expression and purification of outer membrane secretin PilQ[406-770 of Neisseria meningitidis serogroup B

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B, since cross-reactivity of the serogroup B capsule with human tissue has hampered efforts to develop a reliable vaccine. PilQ is an antigenically conserved outer membrane protein which is essential for meningococcal pilus expression at the cell surface. In the current study, we selected a 1095bp fragment of C-terminal of secretin pilQ and evaluated the immunogenicity of this recombinant fragment. This fragment was amplified by PCR from genomic DNA isolated from N. meningitidis serogroup B and cloned into the pET-28a expression vector. PilQ406-770 was over-expressed with IPTG and then affinity-purified by Ni2+-Sepharose resin. The recombinant PilQ406-770 was reacted with rabbit anti-N. meningitidis polyclonal antibody in western-blot analysis. Mice were immunized subcutaneously with purified rPilQ[406-770] mixed with an equal volume of Freud's adjuvant and evaluated specific serum antibody responses. Our results show pilQ[406-770] cloned in pET28a vector, while the cloning of pilQ[406-770] was confirmed by colony-PCR and enzymatic digestion. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET28a- pilQ406-770-BL21 efficiently produces target recombinant protein with molecular weight of 43 kDa in the form of dissoluble inclusion body. Our results confirmed that a prokaryotic expression system for PilQ406-770 protein was successfully constructed

[Study on co-administration of GM-CSF, IL-15 on the enhancement of the immunogenicity of HIV-1 P24-Nef expression vector in BALB/c mouse model]

Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct

[Evaluation of immune responses from immunization of HIV-1 P24-Nef fusion peptide vaccine with monophosphoryl lipid A [MPL] in BALB/c mice]

Several vaccines against HIV have been investigated but none has been approved as an effective HIV vaccine. An approach that could induce stronger immune response against the pathogen is utilizing a multi-epitopic vaccine. This strategy was used in the design of several vaccines and resulted in improved immune responses. In this study a multi-epitopic fusion peptide including parts of HIV-1 Nef and P24 as a vaccine candidate was injected into mice and immune humoral responses measured with total antibody and IgG sub-classes using ELISA. Also measurement of cellular immune responses through evaluation of spleen cells proliferation response using MTT and cytotoxicity by LDH were performed. Finally, the cytokine pattern of IFN-gamma and IL-4 were also determined with ELISA. The results indicate that candidate vaccine stimulated mouse splenic lymphocyte proliferation response and also induced strong cytotoxicity responses. Analysis of humoral immune response has shown that the candidate vaccine has induced specific antibody production mainly of the IgG2a sub-class. Also cytokine pattern evaluation has shown that IFN-gamma secretion was dominant. The use of immunogen and conserved epitopes from P24 and Nef induced strong humoral and cellular immune responses and this construct could be candidate for further studies in animal models

[In vitro expansion of antigen-specific natural regulatory T cells in hepatitis C virus infection mediated by Core antigen]

Hepatitis C virus [HCV] is one of the most relevant persistent infections afflicting the human population. Control of viral replication in HCV infection has been associated with the cellular component of the host immune response. Several mechanisms have been proposed to explain this abnormal immune response, among them an altered activity of regulatory T cells [Tregs] being the most recently postulated. As the first report, in the present study the ability of HCV-core antigen in increase the frequency of natural Tregs [nTregs] in the mixed population of PBMCs was evaluated. Peripheral blood mononuclear cells [PBMCs] from chronic HCV infected patients [n=19] and normal controls [n=6] were analyzed to study the effect of HCV-core antigen in frequency of HCV specific nTregs. For this, PBMCs of different groups were isolated, cultured and stimulated with core antigen. Then an in-house triple-stain flowcytometric method was used to investigate the frequency of nTregs. The results showed that, following incubation with HCV-core Ag, a population of nTregs was increased but, in negative controls the number of nTregs did not increase. The present data supporting the idea that nTregs are able to respond specifically to HCV antigen suggests that Tregs could contribute to an inadequate response against the HCV infection, leading to chronic infection and supports the view that specific natural Tregs may be implicated in host immune tolerance during HCV infection. It is reasonable that HCV vaccine candidates avoid epitopes that lead to strong nTregs stimulation

Candida albicans structural and secreted proteins modulate CD4/CD8 ratio in tumor infiltrating lymphocytes of spontaneous adenocarcinoma bearing mice

Candida albicans is one of the most important opportunistic pathogens that suppress immunologic mechanisms of the host. It is speculated that structural and secretory proteins of C. albicans have immunomodulatory effects in cancer. To evaluate the effects of C. albicans structural and secreted proteins on intratumoral CD4/CD8 ratio as well as the survival rate in BALB/c tumor model. Structural and secretory proteins from C. albicans were isolated and examined for their effects on tumor growth and survival of adenocarcinoma bearing mice. The results indicated that in mice treated with C. albicans structural protein, the survival rate significantly decreased compared with the control groups. Also, mice treated with secretory proteins showed a decrease in survival rate but it was not statistically significant [p>0.05]. Investigating the frequency of tumor infiltrated CD4+ and CD8+ T lymphocytes indicated that the percentages of tumor infiltrated CD4+ T lymphocytes in response to structural and secreted proteins were higher compared to the control groups. Our study suggests that C. albicans structural and secreted proteins modulate intratumor T lymphocyte infiltration

Immunogenicity of a new HIV-1 DNA Construct in a BALB/c mouse model

Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progression. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. In the present study a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. HIV-1 P24-Nef gene was cloned in PCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-gamma evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-gamma [p<0.05]. Cytotoxicity and lymphocyte proliferation responses of mice vaccinated with the candidate vaccine were significantly increased compared to control groups [p<0.05]. HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model

Effect of invasive aspergillosis infection on the immune responses of Cancer mice

Using a cancer murine model of invasive aspergillosis [IA], we investigated the expression of TLR-2, Dectin-1 and the level of cytokine production by CD4+ T helper cells in different groups of mice [with or without cancer], also, the effect of invasive aspergillosis on the immune response pattern of cancer mice. Patterns of susceptibility and resistance to infection obtained with different groups of mice injected with Aspergillus fumigatus conidia. TLR-2 and Dectin-1 analyzed applying flowcytometry and cytokine production of cultured splenocytes by ELISA method. Cancer mice that challenged with A. fumigatus conidia showed significant increase in TLR-2 and Dectin-1 levels compared with the two other control groups [normal mice challenged with A. fumigatus and non-infected cancer mice]. Moreover, it showed insignificant decrease in IFN-gamma and IL-10 levels and insignificant increase in TNF-alpha level. The data demonstrated remarkable rise in IL-4 level and the mortality of cancer mice that intravenously infected with A. fumigatus. Probably IA causes stimulation in innate immunity and Th2 cells, also some disorganization in cytokine production in CD4+ T helper cells. We hypothesize that concomitance of IA and cancer may change the microenvironment for local or systemic immune responses. Other complementary studies could help supporting our hypothesis

Immune response analysis of immunized Balb/C mice with HIV-1 fusion p24 - gp41 genes as DNA vaccine candidate

The global HIV epidemic continues to expand and exceeding previous predictions. An effective vaccine represents the best hope to curtail the HIV epidemic. DNA vaccines induce humoral and cellular responses and mimic live vaccines without their pathogenic potential. The importance of CD8[+]CTL responses in controlling HIV and SIV viremia has led to production of a series of vaccines candidates that effectively induce these responses. It is now widely believed that an HIV vaccine strategy must stimulate both a strong humoral [antibody] as well as cell-mediated [CTL] immune response. The p24 and gp41 play many important roles in host-virus interaction and pathogenesis. These proteins are considered as attractive vaccine candidate in which their immunogenecity and immunomodulatory effects have been confirmed. In this study, a construct, pcDNA3.1Hygro- [p24-gp41], was evaluated as a DNA vaccine candidate in Balb/C mice for generation of effective cellular immune responses. For immunizing, we used dendrosome, a novel family of vehicles for transfection and therapy. IFN-gamma cytokine production and total antibody were detected by ELISA. Lymphoprolifration assay was performed by MTT test. ELISA and MTT assays confirmed that the cited p24-gp41 fusion gene is able to enhance immune responses in mice. The construct that was used in this research can be a good candidate for DNA vaccine against HIV-1, if the future complementary tests demonstrate the same trends of immunogenic responses shown in this study