Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types <it>in vitro</it>. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

Skin Electroporation: Effects on Transgene Expression, DNA Persistence and Local Tissue Environment

BACKGROUND: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. CONCLUSIONS/SIGNIFICANCE: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance

Prime-boost immunization strategies against HIV-1

Since the start of the global HIV epidemic in 1981, more than twentyfive million people have succumbed to AIDS-related diseases. The epidemic is most severe in Sub-Saharan Africa, where twentyfive million people are currently infected and more than two million died during 2006. An indication of the epidemic s severity is that every hour the virus infects between 300 and 800 people. It is clear that an effective prophylactic vaccine is desperately needed. Attempts to utilize classical vaccine strategies against HIV-1 have proven inefficient or potentially harmful and researchers have therefore been obliged to explore novel vaccine approaches. Genetic immunization is a new way of inducing immune responses against antigens deriving from microbial pathogens or tumors. The gene encoding the antigen of interest is introduced into the body by means of an expression vector, which commonly is a recombinant bacterial plasmid or an attenuated recombinant microbe. This method of immunization has the primary benefit of inducing an immune response that mimics the response to natural infection with an intracellular pathogen. The primary strategy we use to induce high levels of broadly reactive immune responses against HIV-1 is immunization with naked DNA encoding multiple viral antigens in combination with potent adjuvants. The immune responses that are induced against plasmid-encoded antigens can be significantly augmented by subsequently boosting with additional vaccine modalities, an immunization protocol referred to as heterologous prime boost. Specifically, we show in mice that it is possible to obtain both humoral and cellular immune responses to all plasmid-encoded HIV-1 antigens in a multi-plasmid vaccine. The cytokine granulocyte macrophage-colony stimulating factor acted as an adjuvant. We also show that spatial separation of the vaccine components could augment the immune responses to some of the included antigens. Further, the responses induced by the multi-plasmid vaccine were efficiently boosted using Modified Vaccinia virus Ankara (MVA) carrying similar, but not identical, HIV-1 genes. The MVA boost resulted in significantly increased levels of HIV-1 specific antibodies as well as extremely high levels of polyfunctional CD8+ T cell responses directed against all included HIV-1 antigens. We also show the capacity of DNA and MVA to induce long-lived vaccine-specific immunological memory. Importantly, DNA was shown capable of efficiently boosting an immune response primed almost one year earlier by the same DNA construct. Moreover, the capacity of different vaccine protocols to induce protection against a cell-based HIV-1 challenge was demonstrated in an experimental model. Protection against challenge was obtained by immunizing with plasmids encoding HIV-1 Gag, Env and Tat, either alone or by priming with the plasmids and subsequently boosting with the corresponding proteins. With one exception, all the vaccine constructs described here have been or are currently being evaluated in clinical trials. In our initial phase I trial, the multigene/multisubtype vaccine has shown great potential to induce HIV-1 specific cellular immune responses in humans that can be dramatically augmented and broadened by boosting with the recombinant MVA. This vaccine protocol is currently being evaluated in a phase I trial in Tanzania. Overall, the preclinical data presented in this thesis have translated well into immunogenicity in clinical trials

Absence of Nosocomial Transmission of Imported Lassa Fever during Use of Standard Barrier Nursing Methods

Nosocomial transmission of Lassa virus (LASV) is reported to be low when care for the index patient includes proper barrier nursing methods. We investigated whether asymptomatic LASV infection occurred in healthcare workers who used standard barrier nursing methods during the first 15 days of caring for a patient with Lassa fever in Sweden. Of 76 persons who were defined as having been potentially exposed to LASV, 53 provided blood samples for detection of LASV IgG. These persons also responded to a detailed questionnaire to evaluate exposure to different body fluids from the index patient. LASV-specific IgG was not detected in any of the 53 persons. Five of 53 persons had not been using proper barrier nursing methods. Our results strengthen the argument for a low risk of secondary transmission of LASV in humans when standard barrier nursing methods are used and the patient has only mild symptoms

Long-Lasting Mucosal and Systemic Immunity against Influenza A Virus Is Significantly Prolonged and Protective by Nasal Whole Influenza Immunization with Mucosal Adjuvant N3 and DNA-Plasmid Expressing Flagellin in Aging In- and Outbred Mice

Background: Vaccination is commonly used to prevent and control influenza infection in humans. However, improvements in the ease of delivery and strength of immunogenicity could markedly improve herd immunity. The aim of this pre-clinical study is to test the potential improvements to existing intranasal delivery of formalin-inactivated whole Influenza A vaccines (WIV) by formulation with a cationic lipid-based adjuvant (N3). Additionally, we combined WIV and N3 with a DNA-encoded TLR5 agonist secreted flagellin (pFliC(-gly)) as an adjuvant, as this adjuvant has previously been shown to improve the effectiveness of plasmid-encoded DNA antigens.  Methods: Outbred and inbred mouse strains were intranasally immunized with unadjuvanted WIV A/H1N1/SI 2006 or WIV that was formulated with N3 alone. Additional groups were immunized with WIV and N3 adjuvant combined with pFliC(-gly). Homo and heterotypic humoral anti-WIV immune responses were assayed from serum and lung by ELISA and hemagglutination inhibition assay. Homo and heterotypic cellular immune responses to WIV and Influenza A NP were also determined.  Results: WIV combined with N3 lipid adjuvant the pFliC(-gly) significantly increased homotypic influenza specific serum antibody responses (&gt;200-fold), increased the IgG2 responses, indicating a mixed Th1/Th2-type immunity, and increased the HAI-titer (&gt;100-fold). Enhanced cell-mediated IFNγ secreting influenza directed CD4+ and CD8+ T cell responses (&gt;40-fold) to homotypic and heterosubtypic influenza A virus and peptides. Long-term and protective immunity was obtained.  Conclusions: These results indicate that inactivated influenza virus that was formulated with N3 cationic adjuvant significantly enhanced broad systemic and mucosal influenza specific immune responses. These responses were broadened and further increased by incorporating DNA plasmids encoding FliC from S. typhimurum as an adjuvant providing long lasting protection against heterologous Influenza A/H1N1/CA09pdm virus challenge. View Full-TextFunding Agencies|VinnovaVinnova [D09-01, Halsofonden 09-4]</p

Maternal immune status influences HIV-specific immune responses in pups after DNA prime protein boost using mucosal adjuvant

This study was designed to determine the impact of maternal HIV-1 specific immunity on HIV-DNA immunization of 2 weeks old pups during the breast-feeding period. Adult female mice received intranasal or intradermal HIV DNA (gp160Env, p37Gag, Nef, Tat and Rev) prime and recombinant protein booster immunizations that induced mucosal and systemic HIV-1 specific B and T cell responses. Intranasal administration of the immunogens induced higher serum IgG titers to HIV antigens than the intradermal immunization. Furthermore, predominantly higher HIV-1 specific fecal IgA titers were obtained in nasally immunized mice. The capacity to respond to one single prime with DNA and one boost with recombinant protein was then compared in pups born to mothers displaying HIV-1-specific immune responses and in pups born to non-vaccinated mothers. Immune responses to the greatest number of antigens were detected in pups born to mothers having the highest HIV-1 specific immune responses. These data suggest that HIV-1 DNA-plasmid immunization during breast-feeding and recombinant protein boosting shortly thereafter enhances the breadth of the humoral HIV-1 specific immune responses.Original publication: Andreas Bråve, Kari Johansen, Paolo Palma, Reinhold Benthin and Jorma Hinkula, Maternal immune status influences HIV-specific immune responses in pups after DNA prime protein boost using mucosal adjuvant, 2008, Vaccine, (26), 47, 5957-5966. Copyright: Elsevier B.V., http://www.elsevier.com

Oral delivery of transgenic plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to higher doses

Background The gut associated lymphoid tissue (GALT) includes around two thirds of the total lymphoid system. CD4+ T-cells in the GALT are a main target for HIV during primary infection. Thus, immunization targetting GALT is likely to be of importance for an effective vaccine strategy. Transgenic plants expressing HIV antigens can reach GALT conveniently. This system allows multiple boosts, has simple logistics (no cold chain, no injections) and large production capacity. Methods Three groups of mice were given extract from plant lines expressing HIV-1 p24 at (A) low level (20 ng/feeding); (B) high level (460 ng/feeding); (C) control (wild type, 0 ng). No adjuvant was included. The extracts were administered by gastric tube day 0, 14 and 28. On day 55 all mice were given an intramuscular (i.m.) boost with 10 micrograms of purified p24 antigen. Immune responses were determined by measurement of p24-antibodies in serum by ELISA. Results The mice immunized by the low dose plant line (A) showed a higher systemic immune response after i.m. boost compared to the high dose group (B). The w.t. controls (C) had undetectable p24-responses. The responses in group A were 3 to 10 times higher (ELISA OD values) than in group B. Pre-boost antibody responses were at background levels in all groups. Preliminary analyses indicate a predomninant Th1-type response (antigen-specific IgG2a higher than IgG1). Conclusion Simple and inexpensive means of vaccination are important in order to reach large numbers of people with effective vaccine regimens. The HIV-1 p24 low dose transgenic plant extracts given orally showed a superior priming effect in mice compared to the p24 high dose extracts. This could be an immunization method and route worth exploring further.Molekylärt jordbrukMolekylär biokemiUtveckling av slemhinneadministrerade vaccine

IL-2 release from splenocytes, stimulated with different antigens.

<p>Mice were immunized with a split influenza vaccine (Vaxigrip) containing the A/H1N1/Brisbane/2007 strain with or without adjuvant. The groups were immunized three times with three-week intervals. Splenocytes were analyzed regarding IL-2 release after influenza stimuli. (A) IL-2 responses against influenza A/H1N1/Brisbane/59/2007 (whole virus). (B) IL-2 responses against influenza A/H1N1/ California/04/2009 (whole virus). (C) IL-2 responses against influenza nucleoprotein-peptides from A/H1N1/Brisbane. Median and range is shown for each group and statistical significance compared to the non-adjuvanted group are indicated, *p<0.05, **p<0.01 and ***p<0, 001.</p