MALDI imaging of post‐mortem human spinal cord in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressing disease of the central nervous system that is characterized by motor neuron degeneration in the brainstem and the spinal cord. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry is an emerging powerful technique that allows for spatially resolved, comprehensive, and specific characterization of molecular species in situ. In this study, we report for the first time the MALDI imaging‐based spatial protein profiling and relative quantification of post‐mortem human spinal cord samples obtained from ALS patients and controls. In normal spinal cord, protein distribution patterns were well in line with histological features. For example, thymosin beta 4, ubiquitin, histone proteins, acyl‐CoA‐binding protein, and macrophage inhibitory factor were predominantly localized to the gray matter. Furthermore, unsupervised statistics revealed a significant reduction of two protein species in ALS gray matter. One of these proteins (m/z 8451) corresponds to an endogenous truncated form of ubiquitin (Ubc 1–76), with both C‐terminal glycine residues removed (Ubc‐T/Ubc 1–74). This region‐specific ubiquitin processing suggests a disease‐related change in protease activity. These results highlight the importance of MALDI mass spectrometry as a versatile approach to elucidate molecular mechanisms of neurodegenerative diseases.     

Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P_cy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4⁺ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4⁺ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.     

Multiprotein genetic vaccine in the SIV-Macaca animal model: a promising approach to generate sterilizing immunity to HIV infection

BACKGROUND: Vaccine combining structural and regulatory proteins is an emerging approach to develop an HIV/AIDS vaccine and therefore, the immunogenicity and efficacy of two regimens of immunization combining structural (Gag/Pol, Env) and regulatory (Rev, Tat, Nef) Simian immunodeficiency virus (SIV) proteins were compared in cynomolgus monkeys. METHODS: Monkeys were immunized with Modified Vaccine Ankara vector (MVA-J5) (protocol 1) or with DNA, Semliki forest virus and MVA vectors (DNA/SFV/MVA) (protocol 2). At week 32, all monkeys were challenge intravenously (protocol 1) or intrarectally (protocol 2) with 50 MID(50) of SIVmac251. Humoral, proliferative responses and in particular in protocol 2, the frequency of IFN-gamma producing cells, were measured in all monkeys before and after the challenge. RESULTS: Both vaccine regimens elicited humoral and proliferative responses but failed to induce neutralizing antibodies. Upon intravenous challenge, two out of three MVA-J5 vaccinated monkeys exhibited a long-term control of the viral replication whereas DNA/SFV/MVA vaccine abrogated the virus replication up to undetectable level in three out of four vaccinated monkeys. A major contribution to this vaccine effect appeared to be the IFN-gamma/ELISPOT responses to vaccine antigens (Gag, Rev Tat and Nef). CONCLUSIONS: These results, indicate that multiprotein heterologous prime-boost vaccination can induce a robust vaccine-induced immunity able to abrogate virus replication.     

Study of immunological and virological parameters during thalidomide treatment of SIV-infected cynomolgus monkeys

The potential therapeutic utility of thalidomide (Thd), an effective inhibitor of tumor necrosis factor (TNF)-α in vitro , was investigated in cynomolgus monkeys ( Macaca fascicularis ) at 10 months after infection with simian immunodeficiency virus (SIV). Thd-treated macaques (n=8) received an oral dose (10 mg) daily for 7 days, followed by a wash-out period of 5 weeks. A 2nd cycle of treatment was performed on the same animals at higher doses (20 mg Thd/day) for 14 days. The control monkeys (n=7) received a placebo for the same period of time. In the present study, we show that Thd, in addition to inhibiting TNF-α production after in vitro mitogen stimulation of peripheral blood mononuclear cells (PBMCs), was able to restore the proliferative responses to SIV peptides in monkeys that were infected with SIV. Interestingly, we found that such effects are associated with an increased expression of CD28 cell surface receptors on CD4⁺ T-cells paralleled by a decrease on CD8⁺ T-cells. At the same time, significant reduction in either cell-associated viral load or plasma viral RNA was not observed among the SIV-infected monkeys during the two treatment cycles, when compared with the placebo group.     

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine.

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.     

Effect of MHC Haplotype on Immune Response upon Experimental SHIVSF162P4cy Infection of Mauritian Cynomolgus Macaques

Little is known about the effects of Major Histocompatibility Complex (MHC) haplotypes on immunity to primate lentiviruses involving both acquired and innate immune responses. We present statistical evidence of the influence of MHC polymorphism on antiviral immunity of Mauritian cynomolgus macaques (MCM) following simian/human immunodeficiency virus SHIV_SF162P4cy infection, involving the production of pro- and anti-inflammatory cytokines and α-defensins, which may modulate acquired immune responses. During the acute phase of infection, IL-10 correlated positively with viral load and negatively with CD4+T cell counts. Furthermore, α-defensins production was directly correlated with plasma viral RNA, particularly at peak of viral load. When the effects of the MHC were analyzed, a significant association between lower anti-Env binding and neutralizing antibody levels with class IB M4 haplotype and with class IA, IB M4 haplotype, respectively, was observed in the post-acute phase. Lower antibody responses may have resulted into a poor control of infection thus explaining the previously reported lower CD4 T cell counts in these monkeys. Class II M3 haplotype displayed significantly lower acute and post-acute IL-10 levels. In addition, significantly lower levels of α-defensins were detected in class IA M3 haplotype monkeys than in non-M3 macaques, in the post-acute phase of infection. These data indicate that the MHC could contribute to the delicate balance of pro-inflammatory mechanisms, particularly with regard to the association between IL-10 and α-defensins in lentivirus infection. Our results show that host genetic background, virological and immunological parameters should be considered for the design and interpretation of HIV-1 vaccine efficacy studies.     

Immunization of Macaca fascicularis with inactivated SIV preparations: Challenge with human- or monkey-derived SIV and the effects of a longer immunization schedule

In cynomolgus monkeys, we compared two human-derived SIV_mac251 whole virus vaccines, a long vs short immunization schedule, and two different challenge viruses. Both vaccines induced protection after challenge with human-derived SIV_mac251/32H. There was no difference between the two schedules of immunization. Seven monkeys, five of which were protected following the first challenge, were reboosted and rechallenged with monkey-derived SIV_mac251, but no protection was observed. The titers of anti-human cell or -SIV neutralizing antibodies were not related to protection.     

Vaccination of pregnant cynomolgus monkeys with whole formalin-inactivated SIVmac251.

Five pregnant (two to three and one-half months) Macaca fascicularis seroconverted following immunization with sucrose-gradient purified and formalin-inactivated whole simian immunodeficiency virus (SIVmac251). No untoward effects on fetal maturation were observed during the immunization of the mothers. Antibodies to SIVmac251 (also those with in vitro neutralizing activity) were passively transferred to the offspring but disappeared within two to six months after birth. Antibodies to env glycoprotein (gp130) lasted longer than those against viral gag proteins (p26,p60).     

Anti-nef antibodies and other predictors of disease progression in HIV-1 seropositive injecting drug users.

A cross-sectional and retrospective longitudinal study has been conducted in three Italian infectious disease centres to evaluate the role of anti-nef antibodies and other markers (HIV-1 p24 antigen, p24 Ag; Beta 2-microglobulin, B2-M; and number of CD4+ lymphocytes) as predictors of disease progression in HIV seropositive injecting drug users (IDUs). The selected patients were: 1) HIV-seropositive IDUs in different stages of HIV infection; 2) HIV-seropositive IDUs who had developed AIDS, from whom serial serum samples were available during the asymptomatic stage, and 3) HIV seropositive IDUs who remained asymptomatic through a follow-up period of the same duration as the patients who developed AIDS. Absence of anti-nef antibodies was associated with symptomatic HIV infection. A significant association between the absence of anti-nef antibodies, the presence of p24 Ag, high levels of B2-M, a number of CD4+ lymphocytes less than 500/ml at first visit and disease progression was found. Subjects who were persistently positive for antibody to nef were less likely to develop AIDS than those who were transiently or persistently negative. This difference was statistically significant (p = 0.03). The results of this study show that absence or disappearance of anti-nef antibodies may be used as predictor of disease evolution in HIV seropositive IDUs. This study also confirms the usefulness of other markers, such as p24 Ag, B2-M and number of CD4+ lymphocytes previously shown to be predictive of rapid disease progression for predicting the course of HIV seropositive IDUs.