There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-␥)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2 d ) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2 b ) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-␥. Tuberculosis (TB), an airborne disease caused by Mycobacterium tuberculosis, is responsible for 2 million deaths each year, with more than 90% of cases occurring in developing countries. It has been estimated that one-third of the world population is infected with M. tuberculosis, and about 5 to 10% of the infected individuals will develop TB during their lifetime. The increasing global impact of TB has been linked to growing poverty, increased emigration, deterioration of public health care, the spread of human immunodeficiency virus, and the development of multidrug-resistant strains of M. tuberculosis. Bacille Calmette-Guérin (BCG), a live and attenuated strain of Mycobacterium bovis, is the only available vaccine against TB to date and has been used for the vaccination of newborns for decades. The vaccine is effective in preventing serious complications of childhood TB, but its efficacy wanes over a period of 10 to 15 years, rendering adolescents increasingly susceptible to pulmonary TB. Significant efforts are being made to generate a more effective TB vaccine (3,24,50), and vaccination regimens aimed to improve BCG-induced protection are currently the most favorable approach (4,14,19,21,29,36). The most promising antigens for vaccine generation include protei...
A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (⌬E1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV ؉ ) individuals is as low as that among HIV ؊ individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.Adenovirus vectors are being developed for gene therapy purposes with the aim to treat inherited or acquired disease (6, 10, 15, 21) as well as for therapeutic and prophylactic vaccination strategies (5, 32). The use of adenovirus for vaccination has recently been fueled by highly promising results demonstrating protection against viruses and viral diseases in rodents and nonhuman primates (28,34,35) as well as induction of Tand B-cell responses in humans in early phase I vaccine trials with healthy volunteers (7). However, high seroprevalence and high neutralizing antibody (NAb) titers against the commonly used vectors, i.e., adenovirus type 5 (Ad5) and Ad2 (39), hamper the application of C group-based vectors, since circulating NAbs efficiently capture administered recombinant vectors obscuring therapeutic effect (19). It has been shown, at least in rodents (1, 42), nonhuman primates (3), and humans in early phase I clinical trials (7), that high levels of NAbs decrease gene transfer efficiency or blunt vaccine potency. Since levels of NAbs vary among individuals, overdosing with recombinant vector in an attempt to overcome the neutralizing activity may result in either excellent clinical results or severe vector-mediated toxicity. Thus, the presence of anti-Ad5 preexisting immunity does not allow accurate dose control and thereby may prevent the universal use of Ad5-based vectors as gene transfer vehicles in humans.Although many strategies are being pursued to avoid vector neutralization (3,5,19,23,41,43), a most viable strategy is the use of rare human adenovirus...
Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T-and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (>5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8 + T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.
The most advanced malaria vaccine, RTS,S, is comprised of an adjuvant portion of the Plasmodium falciparum circumsporozoite (CS) protein fused to and admixed with the hepatitis B virus surface antigen. This vaccine confers short-term protection against malaria infection, with an efficacy of about 50%, and induces particularly B-cell and CD4؉ T-cell responses. In the present study, we tested by the hypothesis that the Th1 immune response to CS protein, in particular the CD8 ؉ T-cell response, which is needed for strong and lasting malaria immunity, is boosted to sustainable levels vectors adenovirus and 26 with an homologous insert 35 (Ad35.CS/Ad26.CS). In this study, we evaluated immune responses induced with vaccination regimens based on an adjuvant-containing, yeast-produced complete CS protein followed by two recombinant low-seroprevalence adenoviruses expressing P. falciparum CS antigen, Ad35.CS (subgroup B) and Ad26.CS (subgroup D). Our results show that (i) the yeast (Hansenula polymorpha)produced, adjuvanted full-length CS protein is highly potent in inducing high CS-specific humoral responses in mice but produces poor T-cell responses, (ii) the Ad35.CS vector boosts the gamma interferon-positive (IFN-␥
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