Reduced vitamin D-induced cathelicidin production and killing of Mycobacterium tuberculosis in macrophages from a patient with a non-functional vitamin D receptor: A case report.

Tuberculosis (TB) presents a serious health problem with approximately a quarter of the world's population infected with Mycobacterium tuberculosis (M. tuberculosis) in an asymptomatic latent state of which 5-10% develops active TB at some point in their lives. The antimicrobial protein cathelicidin has broad antimicrobial activity towards viruses and bacteria including M. tuberculosis. Vitamin D increases the expression of cathelicidin in many cell types including macrophages, and it has been suggested that the vitamin D-mediated antimicrobial activity against M. tuberculosis is dependent on the induction of cathelicidin. However, unraveling the immunoregulatory effects of vitamin D in humans is hampered by the lack of suitable experimental models. We have previously described a family in which members suffer from hereditary vitamin D-resistant rickets (HVDRR). The family carry a mutation in the DNA-binding domain of the vitamin D receptor (VDR). This mutation leads to a non-functional VDR, meaning that vitamin D cannot exert its effect in family members homozygous for the mutation. Studies of HVDRR patients open unique possibilities to gain insight in the immunoregulatory roles of vitamin D in humans. Here we describe the impaired ability of macrophages to produce cathelicidin in a HVDRR patient, who in her adolescence suffered from extrapulmonary TB. The present case is a rare experiment of nature, which illustrates the importance of vitamin D in the pathophysiology of combating M. tuberculosis.

Interleukin-23 instructs protective multifunctional CD4 T cell responses after immunization with the Mycobacterium tuberculosis subunit vaccine H1 DDA/TDB independently of interleukin-17A.

Interleukin (IL)-17A-producing T helper (Th)17 cells are increasingly being acknowledged to be associated with protective immunity to Mycobacterium tuberculosis (Mtb). Subunit vaccines potently promote protective immune responses against Mtb infection that correlate with an expansion of IL-23-dependent Th17 cells. Previous studies revealed that after vaccination, IL-23 is required for protection against challenge with Mtb but the underlying IL-23-dependent-and possibly IL-17A-mediated-mechanisms remain elusive. Therefore, we here analyzed the early outcome of Mtb infection in C57BL/6, IL-23p19-deficient (-/-), and IL-17A-/- mice after vaccination with the subunit vaccine H1-DDA/TDB to investigate the role of the IL-23-Th17 immune axis for the instruction of vaccine-induced protection. While in IL-23p19-/- mice the protective effect was reduced, protection after vaccination was maintained in IL-17A-/- animals for the course of infection of 6 weeks, indicating that after vaccination with H1-DDA/TDB early protection against Mtb is-although dependent on IL-23-not mediated by IL-17A. In contrast, IL-17A deficiency appears to have an impact on maintaining long-term protection. In fact, IL-23 instructed the vaccine-induced memory immunity in the lung, in particular the sustained expansion of tumor necrosis factor (TNF)+IL-2+ multifunctional T cells, independently of IL-17A. Altogether, a targeted induction of IL-23 during vaccination against Mtb might improve the magnitude and quality of vaccine-induced memory immune responses. KEY MESSAGES: After subunit Mtb vaccination with H1-DDA/TDB, IL-23 but not IL-17A contributes to vaccine-induced early protection against infection with Mtb. IL-17F does not compensate for IL-17A deficiency in terms of H1-DDA/TDB-induced protection against Mtb infection. IL 23 promotes the H1-DDA/TDB-induced accumulation of effector memory T cells independently of IL 17A. IL-23 arbitrates the induction of H1-specific IFN-γ-TNF+IL-2+ double-positive multifunctional CD4 T cells after subunit Mtb vaccination in an IL-17A-independent manner.

Boosting BCG with proteins or rAd5 does not enhance protection against tuberculosis in rhesus macaques.

Tuberculosis (TB) is the leading cause of death from infection worldwide. The only approved vaccine, BCG, has variable protective efficacy against pulmonary TB, the transmissible form of the disease. Therefore, improving this efficacy is an urgent priority. This study assessed whether heterologous prime-boost vaccine regimens in which BCG priming is boosted with either (i) protein and adjuvant (M72 plus AS01E or H56 plus CAF01) delivered intramuscularly (IM), or (ii) replication-defective recombinant adenovirus serotype 5 (Ad5) expressing various Mycobacterium tuberculosis (Mtb) antigens (Ad5(TB): M72, ESAT-6/Ag85b, or ESAT-6/Rv1733/Rv2626/RpfD) administered simultaneously by IM and aerosol (AE) routes, could enhance blood- and lung-localized T-cell immunity and improve protection in a nonhuman primate (NHP) model of TB infection. Ad5(TB) vaccines administered by AE/IM routes following BCG priming elicited ~10-30% antigen-specific CD4 and CD8 T-cell multifunctional cytokine responses in bronchoalveolar lavage (BAL) but did not provide additional protection compared to BCG alone. Moreover, AE administration of an Ad5(empty) control vector after BCG priming appeared to diminish protection induced by BCG. Boosting BCG by IM immunization of M72/AS01E or H56:CAF01 elicited ~0.1-0.3% antigen-specific CD4 cytokine responses in blood with only a transient increase of ~0.5-1% in BAL; these vaccine regimens also failed to enhance BCG-induced protection. Taken together, this study shows that boosting BCG with protein/adjuvant or Ad-based vaccines using these antigens, by IM or IM/AE routes, respectively, do not enhance protection against primary infection compared with BCG alone, in the highly susceptible rhesus macaque model of tuberculosis.

Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology.

When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrient-starved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) or IFN-γ, TNF-α, and interleukin-2 to T cells that only expressed IFN-γ. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission.

Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant.

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.

CAF05: cationic liposomes that incorporate synthetic cord factor and poly(I:C) induce CTL immunity and reduce tumor burden in mice.

Considerable effort has been put into targeting tumors through therapeutic vaccination using dendritic cell-, DNA-, protein-, or peptide-based vaccines. Purified peptides and proteins are generally not immunogenic and need to be administered with an adjuvant that will trigger an appropriate immune response. Safe adjuvants that favor induction of tumor reactive CD8(+) T cells with the capacity to directly kill tumor cells are therefore a high priority. We have previously reported on the effect and mechanism of a cationic adjuvant formulation, CAF01, which incorporates synthetic mycobacterial cord factor and primes protective Th1, Th17, and antibody responses in animal models of bacterial, viral, and parasitic infections. The CAF01 adjuvant is currently in clinical trial. Using CAF01 as a backbone, we recently demonstrated that incorporating the TLR3 ligand polyinosinic/polycytidylic acid [poly(I:C)] primes CD8(+) T cells specific to the SIINFEKL epitope of the model antigen ovalbumin. In the present study, we demonstrate that CAF01/poly(I:C), termed cationic adjuvant formulation 05 or CAF05, can induce CD8(+) T cells that efficiently lyse target cells and significantly reduce tumor growth in two different mouse tumor models: lung B16-OVA melanoma expressing ovalbumin and the self-antigen TRP2, and subcutaneous TC-1 tumors expressing the human papillomavirus-16 protein E7.

Enhanced humoral and cell-mediated immune responses after immunization with trivalent influenza vaccine adjuvanted with cationic liposomes.

The recent pandemic caused by new influenza A (H1N1) has emphasized the need for improved influenza vaccines with enhanced immune responses that ideally include longlived humoral and CMI responses and mediate a broad protection. This study demonstrates that administration of trivalent influenza vaccine (TIV) with the cationic liposome adjuvant system CAF01 enhances the humoral immune response as measured by hemagglutinin inhibition titers and influenza-specific serum antibody titers, and promote a strong Th1 response with augmented levels of IL-1ß, IL-2, IL-12, IFN-γ and TNF-α. Furthermore, high levels of IL-17 are detected in agreement with CAF01's ability to promote TH17 responses. Importantly, the Th1/Th17 cytokine profile is still maintained 20 weeks after the last vaccination. The CAF01 adjuvanted influenza vaccine reduces weight loss and temperature decrease and results in complete survival of mice challenged with the drifted H1N1 influenza strain A/PR/8/34. Overall, the results suggest that CAF01 is a potent adjuvant system for future, improved influenza vaccines.