segunda-feira, 28 de fevereiro de 2011

CD4 que faz TNF distingue entre tuberculose latente e doença


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NATURE MEDICINE | LETTER
Dominant TNF-α+ Mycobacterium tuberculosis–specific CD4+ T cell responses discriminate between latent infection and active disease

Alexandre Harari, Virginie Rozot, Felicitas Bellutti Enders, Matthieu Perreau, Jesica Mazza Stalder, Laurent P Nicod, Matthias Cavassini, Thierry Calandra, Catherine Lazor Blanchet, Katia Jaton, Mohamed Faouzi, Cheryl L Day, Willem A Hanekom, Pierre-Alexandre Bart & Giuseppe Pantaleo
AffiliationsContributionsCorresponding author
Nature Medicine (2011) doi:10.1038/nm.2299
Received 09 November 2010 Accepted 05 January 2011 Published online 20 February 2011

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry–based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease.

Main Methods References Acknowledgments Author information Supplementary information
Cellular immunity, particularly of CD4+ T cells, IFN-γ and TNF-α, has a central role in the control of and protection against Mycobacterium tuberculosis (Mtb) infection1, 2. Diagnosis of Mtb infection remains complex and requires several clinical, radiological, histopathological, bacteriological and molecular parameters. IFN-γ release assays measure responses to antigens (for example, 6-kDa early secretory antigenic target (ESAT-6) or 10-kDa culture filtrate antigen (CFP-10)) expressed by Mtb itself and discriminate between infection by Mtb and immunity induced by vaccination with Bacille Calmette-Guérin (BCG)3, 4 but not between active disease and latent infection5, 6.

Studies in the field of antiviral immunity have shown that polyfunctional (IFN-γ+IL-2+TNF-α+) profiles of virus-specific T cell responses, and not IFN-γ production alone, correlated with disease activity7, 8, 9, 10.

Therefore, we have used the same strategy, polychromatic flow cytometry, to functionally characterize Mtb-specific T cells in subjects with latent Mtb infection or active tuberculosis disease and tested the hypothesis that different cytokine profiles of pathogen-specific T cells may discriminate between active tuberculosis disease and latent Mtb infection.

We enrolled an initial cohort of 283 individuals with known diagnosis of Mtb infection in Switzerland and termed it the 'test cohort' (Supplementary Fig. 1). Subjects were selected on the basis of positive IFN-γ ELISPOT responses against CFP-10, ESAT-6 or both. Among the 283 subjects, active tuberculosis disease was diagnosed in 11 subjects on the basis of clinical signs (for example, cough, weight loss and lymphadenopathy), sputum stain for acid-fast bacilli (AFB), culture and PCR for Mtb and chest radiography6 (the Online Methods and Supplementary Table 1 contain detailed clinical parameters). The remaining 272 participants were diagnosed with latent Mtb infection. We first assessed the magnitude of Mtb-specific T cell responses by IFN-γ ELISPOT after stimulation with pools of peptides encompassing CFP-10 or ESAT-6 proteins. In agreement with previous studies11, 12, Mtb-specific T cell responses were similar in subjects with latent infection and active disease (Fig. 1a).

Figure 1: Quantitative and qualitative analysis of Mtb-specific T cell responses in the test cohort.

(a) IFN-γ ELISPOT responses after stimulation with ESAT-6 or CFP-10 peptide pools in a cohort of 283 participants with latent Mtb infection (n = 272) or active tuberculosis disease (n = 11, Supplementary Fig. 1). Shown are the numbers of spot-forming units (SFU) per 106 mononuclear cells. Statistical significance (P values) of the results was calculated by unpaired two-tailed Student's t test using GraphPad Prism 5. Bonferroni's correction for multiples analyses was applied. (b) Qualitative analysis of Mtb-specific CD4+ T cell responses by polychromatic flow cytometry. Shown are representative flow cytometry analyses of the functional profile of Mtb-specific CD4+ T cell responses in participants with either latent Mtb infection (Subject L5, left) or active tuberculosis disease (Subject A2, right). Profiles are gated on live CD3+CD4+ T cells, and the various combinations of IFN-γ, IL-2 and TNF-α are shown following stimulation with ESAT-6 and CFP-10 peptide pools or PPD. NS, not significant; Neg, negative control (unstimulated). (c) Simultaneous analysis of the functional profile of Mtb-specific CD4+ T cells on the basis of IFN-γ, IL-2 or TNF-α production. ESAT-6–, CFP-10– and PPD-specific CD4+ T cell responses are shown (as indicated by the six colored boxes to the right of the panel) from 48 participants with latent Mtb infection and eight participants with active tuberculosis (TB) disease. Representative examples from subject L5 and subject A2 are also identified. All the possible combinations of the various functions are shown on the x axis, whereas the percentages of the distinct cytokine-producing cell subsets within Mtb-specific CD4+ T cells are shown on the y axis. The pie charts summarize the data, and each slice corresponds to the proportion of Mtb-specific CD4+ T cells positive for a certain combination of functions. Colors in the pie charts are indicated by the seven colored boxes at the bottom of the panel. (d) Distribution of CFP-10– and/or ESAT-6–specific CD4+ T cell responses among subjects with latent Mtb infection or active tuberculosis disease.

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We then assessed the functional profile of Mtb-specific T cell responses by polychromatic flow cytometry and a panel of markers including a viability marker and antibodies specific for CD3, CD4, CD8, IL-2, TNF-α and IFN-γ. Owing to blood specimen availability or quality (see flowchart in Supplementary Fig. 1), this analysis was performed in 48 subjects with latent infection and eight subjects with active disease (Supplementary Table 1). Within the group with latent infection, five were investigated for suspected tuberculosis disease but had negative sputum AFB staining and culture and PCR for Mtb. Twenty-three were health-care workers screened for Mtb infection as part of routine surveillance at the Centre Hospitalier Universitaire Vaudois (CHUV; Supplementary Fig. 1). Twenty were investigated for Mtb infection before the initiation of anti-TNF-α antibody treatment and had negative chest radiographs (Supplementary Fig. 1). In agreement with previous studies11, 12, Mtb-specific CD4+ T cell responses in a representative subject with latent Mtb infection (subject L5) were mostly (>70%) polyfunctional (Fig. 1b), that is, producing IFN-γ, IL-2 and TNF-α. In contrast, a representative subject with active tuberculosis disease (subject A2) (Fig. 1b) showed a dominant (>70% of CD4+ T cells) TNF-α–only response. In these two participants, the functional profile of Mtb-specific CD4+ T cells was similar regardless of the stimulus, that is, ESAT-6 or CFP-10 peptide pools or tuberculin purified protein derivative (PPD). Of note, Mtb-specific T cell responses (analyzed by either IFN-γ ELISPOT or flow cytometry) from the 20 subjects analyzed before the initiation of TNF-α–specific antibody treatment were not different from T cell responses in the remaining 28 subjects also diagnosed with latent infection (Supplementary Fig. 2). The marked difference between the functional profile of Mtb-specific CD4+ T cell responses in latent infection versus active disease was confirmed in a total of 142 Mtb-specific CD4+ T cell responses (all P <>90%) also responded to PPD. Of the 142 responses, 21 were detected in subjects with active disease and 121 in subjects with latent infection (Fig. 1c). Of note, we confirmed the differences in the profile of cytokines between active disease and latent infection when we expressed the data as absolute frequency of cytokine-producing CD4+ T cells (Supplementary Fig. 3). The frequency of single-positive TNF-α–producing CD4+ T cells was higher in individuals with active disease (Supplementary Fig. 3).

In summary, in an opportunistically selected (on the basis of a sufficient number of mononuclear cells; Supplementary Data 1) subgroup of individuals subjected to detailed intracellular cytokine staining (ICS) studies, a functional profile (single-positive TNF-α Mtb-specific CD4+ T cells) was associated with disease activity and so might be helpful for rapid diagnosis of active disease as compare to the conventional culture tuberculosis tests, which require up to several weeks.

We then calculated which parameter (that is, functional T cell subset) was the strongest predictive measure of discrimination between active disease and latent infection. For these purposes, because CFP-10 was more frequently recognized than ESAT-6 (Fig. 1d), we focused the analysis on CFP-10–specific CD4+ T cell responses and included ESAT-6–specific CD4+ T cell responses only when CFP-10 responses were negative. We observed the latter scenario in only one individual with active disease and one individual with latent infection (Fig. 1d).

On the basis of the logistic regression analysis of the multiple functionally distinct T cell subsets, the proportion of TNF-α single-positive Mtb-specific CD4+ T cells was the strongest predictive measure of discrimination between active disease and latent infection (area under the curve (AUC) = 0.995 (95% confidence interval: 0.984–1); odds ratio = 1.45; Supplementary Fig. 4). In addition, a cutoff of 37.4% of single-positive TNF-α–producing CD4+ T cells was calculated as the value allowing the best (sensitivity of 100% and specificity of 96%) separation between latent infection and active disease (Supplementary Fig. 4).

A limitation of these results was that the laboratory investigators were not blinded to the diagnosis of latent infection or active disease. We therefore examined peripheral blood mononuclear cells from a second independent cohort termed the 'validation cohort', whose clinical status was unknown to the investigators. We tested whether the proportion of TNF-α single-positive Mtb-specific CD4+ T cells, and particularly the cutoff at 37.4%, could discriminate between latent infection and active disease.

One hundred and fourteen participants from both Switzerland (n = 72) and South Africa (n = 42) were enrolled between 2009 and 2010 to confirm the functional profile also in persons from a setting (South Africa) where tuberculosis is prevalent (Supplementary Fig. 5). Participants from South Africa were enrolled from clinics in the public health sector in Cape Town and Worcester, both in the Western Cape province of South Africa. Participants from Switzerland in the validation cohort were not included in the test cohort described above. Subjects were selected on the basis of the following criteria: positive Mtb-specific IFN-γ ELISPOT responses, absence of Mtb-specific treatment, seronegative for HIV and good general health status (the Online Methods and Supplementary Fig. 5 contain a full description of the subjects). Active tuberculosis disease diagnosis in subjects from both Switzerland and South Africa was based on clinical signs (for example, cough, weight loss and lymphadenopathy), sputum stain for AFB, culture and PCR for Mtb and chest radiography6 (the Online Methods and Supplementary Table 2 contain detailed clinical parameters). Flow cytometry analyses were performed on the 101 subjects from the validation cohort with positive Mtb-specific CD4+ T cell responses (Supplementary Fig. 5).

IFN-γ ELISPOT and CD4+ T cell specific cytokine expression in response to CFP-10, ESAT-6 or both were evaluated, and the data were provided to the biostatistics facility of CHUV. Later, unblinding of the Mtb clinical status allowed us to confirm that IFN-γ ELISPOT responses were not significantly different between latent infection and active disease (Fig. 2a). Of note, the magnitude of Mtb-specific IFN-γ ELISPOT responses (Fig. 2b) and the distribution of CFP-10– and/or ESAT-6–specific CD4+ T cell responses among subjects with latent Mtb infection or active disease were similar between subjects from Switzerland and South Africa (Fig. 2c).

Figure 2: Analysis of Mtb-specific T cell responses in the validation cohort after unblinding of the clinical status.

(a) IFN-γ ELISPOT responses after stimulation with ESAT-6 or CFP-10 peptide pools. Shown are the numbers of SFU per 106 mononuclear cells. Statistical significance (P values) of the results was calculated by unpaired two-tailed Student's t test in GraphPad Prism 5. Bonferroni's correction for multiples analyses was applied. (b) Analysis of Mtb-specific IFN-γ ELISPOT T cell responses in subjects enrolled in Switzerland (CH) and SA. (c) Distribution of CFP-10– and/or ESAT-6–specific CD4+ T cell responses among subjects from the validation cohort with positive and concordant Mtb-specific CD4+ T cell responses (Supplementary Fig. 5).

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With regard to the polychromatic flow cytometric cytokine profile, 15 participants had a dominant TNF-α single-positive Mtb-specific CD4+ T cell response, that is, >37.4%, considered predictive of active disease in the test cohort (Supplementary Fig. 4). After unblinding, active disease was confirmed in 12 of these 15 participants (Fig. 3a). Along the same line, 79 participants had polyfunctional Mtb-specific CD4+ T cells, including a TNF-α single-positive proportion of <37.4%,>37.4% and the other response <37.4%)> 0.05 for positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity), thus providing evidence that the combined analysis of Swiss and South African cohorts is valid. On the basis of the analysis on the combined cohorts, the global performance of the assay was as follows: PPV = 80%, NPV = 92.4%, sensitivity = 66.67% and specificity = 92.41% (Supplementary Fig. 7). Overall, the cytokine profile predicted the clinical diagnosis in 90% of cases. Of note, these values apply to subjects with detectable ICS responses. When subjects with discordant ESAT-6 and CFP-10 responses were also included in the analysis, the correct clinical diagnosis was made in 84% of subjects.

Figure 3: Percentages of CFP-10– or ESAT-6–specific single-positive TNF-α–producing CD4+ T cells of the 94 subjects from the validation cohort with concordant responses against CFP-10 and ESAT-6.

Dashed line represents the cutoff of 37.4% of single-positive TNF-α. (a) Subjects with active disease or latent infection are identified with blue and red dots, respectively. (b) Subjects from South Africa (SA) or Switzerland (CH) are identified with orange and green dots, respectively.

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We then investigated whether the percentage of Mtb-specific TNF-α–producing CD4+ T cells was the parameter with the strongest predictive value of the clinical status in the validation cohort. On the basis of the logistic regression analysis of the multiple functionally distinct T cell subsets, the proportion of TNF-α single-positive Mtb-specific CD4+ T cells was indeed the strongest predictive measure of discrimination between active disease and latent infection (AUC = 0.825 (95% confidence interval: 0.683–0.968); odds ratio = 1.10; Supplementary Fig. 7). In addition, a cutoff of 38.8% (as compared to 37.4% obtained in the test cohort) of single-positive TNF-α–producing CD4+ T cells was calculated as the value allowing the best separation between latent infection and active disease (Supplementary Fig. 7).

We also had the opportunity to analyze T cell cytokine profiles in five participants during untreated active tuberculosis disease and then after tuberculosis treatment (Fig. 4). In agreement with the above data, the percentage of single-positive TNF-α–producing CD4+ T cells was >37.4% in individuals with active tuberculosis disease. We observed a shift to a polyfunctional profile (single-positive TNF-α–producing CD4+ T cells < n =" 283)" n =" 114)">80% purity). Tuberculin Purified Protein Derivative (PPD RT 23) was purchased from Statens Serum Institute.

IFN-γ ELISPOT assays.
ELISPOT assays were performed per the manufacturer's instructions (Becton Dickinson). Briefly, cryopreserved blood mononuclear cells were rested for 8 h at 37 °C, and then 2 × 105 cells were stimulated with peptide pools (1 μg of each single peptide) in 100 μl of complete medium (RPMI + 10%FBS) in quadruplicate conditions as previously described18. Medium only was used as negative control. Staphylococcal enterotoxin B (SEB; Sigma; 200 ng ml−1) was used as a positive control on 50,000 cells. Results are expressed as the mean number of SFU per 106 cells from quadruplicate assays. Only cell samples with >80% viability after thawing were analyzed, and only assays with <50>500 SFU per 106 cells after SEB stimulation were considered as valid. An ELISPOT result was defined as positive if the number of SFUs was ≥55 SFU per 106 cells and more than fourfold higher than the negative control.

Flow cytometry analysis.
For ICS, cryopreserved blood mononuclear cells (1–2 × 106) were rested for 6–8 h and then stimulated overnight in 1 ml of complete medium containing Golgiplug (1 μl ml−1, Becton Dickinson) and CD28-specific antibodies (0.5 μg ml−1, Becton Dickinson) as previously described19. For stimulation of blood mononuclear cells, peptide pools were used at 1 μl ml−1 for each peptide. SEB stimulation (200 ng ml−1) served as positive control. At the end of the stimulation period, cells were stained for dead cells (LIVE/DEAD kit, Invitrogen), permeabilized (Cytofix/Cytoperm, Becton Dickinson) and then stained with antibodies specific for CD3, CD4, CD8, IFN-γ, TNF-α and IL-2. All antibodies but those specific for CD3 (Invitrogen) and CD4 and CD19 (VWR International) were purchased from Becton Dickinson. Cells were then fixed, acquired on an LSRII SORP (four lasers) and analyzed with FlowJo 8.8.2 and SPICE 4.2.3 (developed by M. Roederer, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health) as previously described18. The number of lymphocyte-gated events ranged between 105 and 106 in the flow cytometry experiments shown.

Statistical analyses.
Comparisons of categorical variables were made with Fisher's exact test. Statistical significance (P values) of the magnitude of ELISPOT responses was calculated by unpaired two-tailed Student's t test using GraphPad Prism 5. Bonferroni's correction for multiples analyses was applied. The selection of the optimal parameters to discriminate between cases of latent infection and cases of active disease was performed using a logistic regression analysis followed by a receiver operating characteristic (ROC) curve analysis20, 21, 22 to evaluate the diagnostic performance of each parameter. Results for the optimal parameter (single-positive TNF-α) are summarized as a contingency table giving sensitivity, specificity and positive and negative predictive values (PPV and NPV). Analyses provided include a ROC-curve graph and a sensitivity and specificity graph as a function of the probability cutoff.

References
Acknowledgments
Author information
Main Methods References Acknowledgments Author information Supplementary information
Affiliations
Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Alexandre Harari, Virginie Rozot, Felicitas Bellutti Enders, Matthieu Perreau, Pierre-Alexandre Bart & Giuseppe Pantaleo
Swiss Vaccine Research Institute, Lausanne, Switzerland.
Alexandre Harari & Giuseppe Pantaleo
Division of Pneumology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Jesica Mazza Stalder & Laurent P Nicod
Division of Infectious Diseases, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Matthias Cavassini & Thierry Calandra
Division of Occupational Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Catherine Lazor Blanchet
Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Katia Jaton
Center of Clinical Epidemiology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Mohamed Faouzi
South African Tuberculosis Vaccine Initiative, University of Cape Town, Cape Town, South Africa.
Cheryl L Day & Willem A Hanekom
Contributions
A.H. designed the study, performed the analyses and wrote the manuscript; V.R., F.B.E. and M.P. generated data and performed analyses; J.M.S., L.P.N., M.C., T.C., C.L.B., C.L.D. and W.A.H. recruited study participants; K.J. performed analyses; M.F. performed the statistical analyses; P.-A.B. contributed to the design of the study, performed analyses and wrote the manuscript; G.P. designed the study, supervised the analyses and wrote the paper. All authors have read and approved the final manuscript.

Competing financial interests
The authors declare no competing financial interests.

Corresponding author
Correspondence to: Giuseppe Pantaleo
Supplementary information
Additional data


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sábado, 26 de fevereiro de 2011

Fungos podem ajudar no combate a malária e outras deonças como dengue e leishmaniose


A Fungal 'Vaccine' for Malaria-Carrying Mosquitoes
by Gretchen Vogel on 24 February 2011, 2:00 PM | Permanent Link | 0 Comments

Fungi that attack insects are present in soils worldwide, and they are used in gardens, greenhouses, and open fields to control agricultural pests. In 2005, scientists showed that strains of two different fungi, Beauveria bassiana and Metarhizium anisopliae, could attack the mosquitoes that spread malaria. When the fungal spores come in contact with the mosquito's exoskeleton, they bore their way into the hemolymph—the insect's equivalent of blood—where they grow, ultimately killing the mosquito.

But the fungi take about 2 weeks to kill the insects. "They want to kill slowly, extracting as many nutrients as possible so they can produce more spores," says Raymond St. Leger, an entomologist at the University of Maryland, College Park. Because it takes only 12 to 14 days for the malaria parasite to mature into its infective form inside the mosquito, an insect often has time to spread the parasite before a fungal infection has killed it off, especially if the mosquito is exposed to the fungus several days after it picks up the parasite.

St. Leger and his colleagues originally created a genetically modified version of the fungus M. anisopliae that, thanks to the insertion of an insect-specific neurotoxin, kills mosquitoes more quickly. But a quick kill also has a disadvantage: It can lead to mosquitoes that are resistant to the fungus, because the resistant insects are the only ones who survive long enough to reproduce.

Now St. Leger and his colleagues have engineered strains of M. anisopliae to block the malaria parasite from developing inside the infected mosquito. Online today in Science, they describe inserting different combinations of three different genes into the fungus to block the malaria parasite from entering the mosquito's salivary glands. (Parasites from the salivary glands infect new hosts when an infected mosquito bites a new victim.) One gene codes for a peptide—a short piece of a protein—called SM1 that resembles the parasite protein the parasite uses to enter the salivary glands. The copies of SM1 produced by the fungus block the parasite's way in. Another added gene codes for part of a human antibody that binds to the parasites and causes them to clump together. A third is an antimicrobial protein called scorpine—it was found in scorpions—that kills the malaria parasite.

When the researchers sprayed spores of the genetically modified fungus strains onto mosquitoes in the lab, the spores did not kill the insects faster than the normal fungus did. But they did significantly reduce the number of parasites in the mosquitoes' salivary glands: Six days after receiving spores of the genetically modified strains, the mosquitoes had 98% fewer parasites in their salivary glands than did those treated with normal fungus.

And the genetically modified fungus acts quickly. After giving mosquitoes a malaria-infected blood meal, the researchers waited 11 days before spraying some with normal fungal spores and others with the enhanced spores. Two days later, 86% of fungus-free mosquitoes could transmit malaria, as could 72% of the insects infected with normal fungus, but only 20% of the mosquitoes exposed to the transgenic fungus could do so.

The results are impressive, says Marit Farenhorst, who studies insect-attacking fungi at In2Care, a start-up company in Wageningen, the Netherlands. "In case you are a little bit late in getting the mosquito infected in field, you can still be in time to affect the transmission of malaria," she says. "It's kind of like vaccinating the mosquito." The main drawback of the approach, says Farenhorst, is that fungal spores survive only a few months when applied to walls or other surfaces. It's currently impractical and expensive to have to keep reapplying spores. But several teams are working on better ways to apply the spores and keep them alive, she says.

Although St. Leger acknowledges that widespread suspicion of genetically modified organisms might make it more difficult to persuade people to apply the fungus in their houses, he says the approach is extremely low risk: The fungus strain that the scientists modified infects only mosquitoes, the genes they inserted only recognize human malaria, and the genes are only turned on once the fungus is inside the mosquito. None of the genes give the fungus a survival advantage over the wild strains that are common in soils, he says. Still, St. Leger says, completing relevant safety tests will take several years.

quinta-feira, 24 de fevereiro de 2011

Think of Me

Think of me





Christine:

Think of me, think of me fondly,
When we've said goodbye.
Remember me once in a while -
Please promise me you'll try.
When you find that, once
Again, you long to take your heart back and be free -
If you ever find a moment
Spare a thought for me...
(Transformation to the Gala. Christine is revealed in full costume.)
We never said our love was evergreen,
Or as unchanging as the sea -
But if you can still remember,
Stop and think of me...
Think of all the things
We've shared and seen -
Don't think about the way
things might have been...
Think of me, think of me waking silent and resigned
Imagine me, trying too hard to put you from my mind
Recall those days, look back on all those times
Thinks of the things, we'll never do
There will never be a day, when I won't think of you
Raoul:
Can it be?
Can it be Christine?
Bravo!
Long ago, it's seems so long ago,
how young and inocent we were
She may not remember me,
but I remember her...
Christine:
Flowers fade, the fruits the summer fade,
they have your seasons so do we
but please promise me, that sometimes
you will think...
(Christine vocalizes)
...of me!

http://letras.ms/w2O

quarta-feira, 23 de fevereiro de 2011

Vacina contra HIV protege macacos contra a infecção

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fig 1
















Article
Immunization with HIV-1 gp41 Subunit Virosomes Induces Mucosal Antibodies Protecting Nonhuman Primates against Vaginal SHIV Challenges
Morgane Bomsel1, 2, 3, , , Daniela Tudor1, 2, 3, Anne-Sophie Drillet1, 2, 3, Annette Alfsen1, 2, 3, Yonatan Ganor1, 2, 3, Marie-Gaëlle Roger4, Nicolas Mouz4, Mario Amacker5, Anick Chalifour6, Lorenzo Diomede7, Gilles Devillier8, Zhe Cong9, Qiang Wei9, Hong Gao9, Chuan Qin9, Gui-Bo Yang10, Rinaldo Zurbriggen5, 11, Lucia Lopalco7 and Sylvain Fleury6
1 Mucosal Entry of HIV-1 and Mucosal Immunity, Cell Biology and Host Pathogen Interactions Department, Cochin Institute, CNRS (UMR 8104), 22 rue Méchain, 75014 Paris, France
2 INSERM U1016, 75014 Paris, France
3 Paris Descartes University , 75006 Paris, France
4 PX'Therapeutics, 38040 Grenoble Cedex 9, France
5 Pevion Biotech Ldt, CH-3063 Ittigen/Bern, Switzerland
6 Mymetics Corporation, CH 1066 Epalinges, Switzerland
7 San Rafaele Institute, 20127 Milan, Italy
8 BD Medical-Pharmaceutical Systems, 38801 Le Pont de Claix, France
9 Institute of Laboratory Animal Science, Chaoyang District, Beijing 100021, People's Republic of China
10 National Center for AIDS/STD Control and Prevention, Chinese Center For Disease Control and Prevention, Beijing 100050, People's Republic of China


Summary
Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.
Graphical Abstract


Human immunodeficiency virus (HIV)-1 is mainly acquired at the mucosal site during sexual intercourse. Previous vaccine strategies against HIV-1 were aimed at inducing circulating neutralizing IgG antibodies or cytotoxic T cells (CTLs). Both strategies have repeatedly failed to elicit protection against HIV-1 infection in vivo ([Tatsis et al., 2009], [Rerks-Ngarm et al., 2009] and [Buchbinder et al., 2008]). An alternative strategy could be the development of a vaccine that elicits a mucosal immune response and blocks the entry of the virus at mucosal sites before primary infection takes place locally in the lamina propria.
The advantage of mucosal antibodies as a potential protection mechanism is supported by studies on individuals who are HIV-1-exposed but remain persistently seronegative (HEPS). In these individuals of both genders, one correlate of protection is the presence of gp41-specific IgAs in the blood and genital secretions, which display HIV-1-neutralizing activity and HIV-1 transcytosis-blocking activity ([Devito et al., 2000a], [Miyazawa et al., 2009] and [Tudor et al., 2009]). Transcytosis is one of the mechanisms of entry of the virus into mucosal tissues ([Bomsel, 1997] and [Bomsel et al., 1998]). Gp41-specific IgAs also block in vitro infection of mucosal target cells ([Devito et al., 2000b], [Miyazawa et al., 2009] and [Tudor et al., 2009]), thereby preventing entry into and transcytosis across epithelial cells ([Devito et al., 2000a], [Bomsel et al., 1998] and [Alfsen et al., 2001]) and blocking access of the virus to the lamina propria (Hladik and McElrath, 2008). Although IgGs may interfere with viral infection in tissues underlying mucosal epithelia and secondary lymphoid tissues, mucosal IgAs, which have a known compartmentalized distribution and repertoire, are thought to best protect mucosal surfaces.
Gp41 is the most conserved envelope subunit of HIV-1 with very few potential glycosylation sites as opposed to the surface envelope subunit gp120, whose neutralizing epitopes are widely masked by glycans. Gp41 and especially its hydrophobic membrane proximal external region (MPER), whose structure is strictly dependent on the lipidic environment provided by the viral membrane ([Coutant et al., 2008] and [Sun et al., 2008]), is targeted by the broadly neutralizing IgGs 2F5 and 4E10. These IgGs provide sterilizing immunity against mucosal HIV-1 challenge, as shown by passive transfer studies, even at a low or undetectable concentration of serum antibodies at the time of protection (Hessell et al., 2010). Mucosal IgAs in HEPS individuals also target the extended MPER and exhibit both transcytosis-blocking and infection-neutralizing activity ([Devito et al., 2000a], [Devito et al., 2000b], [Miyazawa et al., 2009] and [Tudor et al., 2009]).
As reviewed by Montero et al. (2008), several immunogens based on peptides and proteins containing the MPER were evaluated in various animal models by monitoring blood IgGs and CTL. HIV-1-neutralizing IgG antibodies could not be elicited in vivo and specific CTL responses, despite an in vitro activity, lacked protective activity in vivo. In these studies, the immunogen design did not properly take into account the 3D structure of viral antigens that most likely differs greatly from its native structure on the virus. Furthermore, the route of immunization was not adapted to induce a humoral mucosal response, either in terms of compartmentalization or epitope specificity (Brandtzaeg 2009).
In this study, we used market-approved virosomes ([Moser et al., 2007] and [Herzog et al., 2009]) as a carrier—in use for humans for more than 10 years—to deliver two distinct HIV-1 gp41 antigens: a recombinant, truncated, trimeric gp41 (rgp41) antigen (Delcroix-Genête et al., 2006) and the P1 peptide ([Alfsen and Bomsel, 2002], [Magérus-Chatinet et al., 2007], [Yu et al., 2008] and [Coutant et al., 2008]). P1 corresponds to the extended MPER region of gp41 and includes the binding site to the mucosal receptor galactosyl-ceramide that is present on epithelial and dendritic cells ([Alfsen and Bomsel, 2002], [Magérus-Chatinet et al., 2007], [Yu et al., 2008] and [Coutant et al., 2008]). We designed the antigens by removing from gp41 the immuno-dominant epitopes (Delcroix-Genête et al., 2006) that are known to be nonneutralizing. Therefore, the current design of the gp41-derived antigens allows for focusing the immune response on protective HIV-1 envelope epitopes. Immunization of nonhuman primate females by both the intramuscular (i.m.) and intranasal (i.n.) routes with both gp41-derived virosome-bound antigens elicited full protection against repeated SHIV-SF162P3 vaginal challenges, whereas immunization by the i.m. route alone elicited protection in just 50% of the animals. The protected animals showed gp41-specific cervicovaginal IgAs and IgGs with transcytosis-blocking and antiviral activities, but showed no neutralizing IgG activity in their serum.
Results
Antigen Design and Vaccine Strategy
The HIV-1 vaccine strategy of this study was designed to focus on the mucosal humoral immune responses against conserved gp41-derived antigens, the P1 peptide (Alfsen and Bomsel, 2002), and a newly engineered trimeric recombinant gp41 (rgp41) (Delcroix-Genête et al., 2006) from the X4 tropic, clade B HXB2 isolate of HIV-1. This new rgp41 (residues 540–664) is a stable trimer, produced in E. coli, that has several conserved key epitopes of gp41 (Serres, 2001; Delcroix-Genête et al., 2006), a deletion of the conserved immunodominant cluster I region, and additional point mutations introduced to break up homology with interleukin-2 (IL-2) and other human proteins. These later deletions were introduced to prevent a potential autoimmune response upon vaccination (Serres, 2001). In addition, rgp41 contains the caveolin binding site-1 (CBD1) shown to induce HIV-1 neutralizing antibodies (Hovanessian et al., 2004) and the QARILAV neutralizing epitope, the latter being targeted by IgAs from HEPS individuals (Miyazawa et al., 2009). The P1 peptide corresponds to the gp41 MPER and contains the galactosyl ceramide-binding site and the 2F5 and 4E10 epitopes. Both the rgp41 antigen and the P1 peptide were linked by the C terminus to phosphatidylethanolamine, allowing their easy insertion into the lipid membrane of virosomes. In this lipidic context, HIV-1 antigens can fold as in situ in the viral membrane ([Coutant et al., 2008] and [Sun et al., 2008]).
The virosome vector is a nonreplicative virus-like particle derived from influenza that is formed by a lipid bilayered vesicle into which molecules of the influenza virus hemagglutinin (HA) and neuraminidase (NA) are inserted, thereby allowing efficient targeting of antigen-presenting cells. Virosomes have intrinsic adjuvant properties, elicit blood and mucosal antibodies (data not shown and Moser et al., 2007), and are licensed for use in human vaccines (Moser et al., 2007). Importantly, pre-existing antibodies against the influenza hemagglutinin (HA) were not reported to prevent revaccination with virosomes in human, in contrast to the situation with some viral vectors (Tatsis et al., 2009).
Female Macaca mulatta of Chinese origin were used to evaluate the in vivo protective efficacy of the gp41-based virosome vaccine against a virulent SHIV vaginal challenge. The mucosal antibody response is highly compartmentalized, at the level of both the induction (immunization route) and the effector sites, in terms of antibody repertoire and specificity (reviewed in Brandtzaeg, 2009). Therefore, the animals were vaccinated by either the intramuscular (i.m.) route of immunization alone, or by a combined intramuscular plus intranasal route (i.m.+i.n.).
Three groups of Chinese rhesus monkeys received four administrations of vaccine at weeks 0, 7, 15, and 23 (Figure 1A). Six animals (control group 1) received the virosome carrier alone. Six animals (group 2) were vaccinated four times by the i.m. route only, whereas six animals (group 3) received two injections by the i.m. route followed by two booster injections by the i.n. route. Each vaccine dose contained 40 μg of P1 and 40 μg of rgp41. One of the animals in group 3 unfortunately died of an unrelated cause before immunization was completed.


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Figure 1.
Immunization and Challenge
As experimental schedule (A), Female Macacca mulatta were immunized four times at week (W) 0, 7, 15, and 23 with a mixture of two distinct virosome formulations corresponding to virosme-P1 and virosome-rgp41 in a total volume of 100 μl per dose. Group 1 received the empty virosome carrier as placebo, whereas groups 2 and 3 received 40 μg of P1 and 40 μg of rgp41 grafted onto virosomes. All animals from groups 1, 2, and 3 were immunized by the i.m route at W0 and W7. Afterward, groups 1 and 2 continued to receive i.m. immunizations at week 15 and 23, whereas animals from group 3 received two i.n. immunizations with 50 μl of the vaccine in each nostril administered with a specific spray device (Accuspray, BD). Blood, vaginal, and rectal washes were collected from each animal at week −4 and 0 for preimmune samples and 1 week after each vaccination event for immune samples (weeks 1, 8, 16, and 24). One month after the last immunization, animals from groups 1 (B), 2 (C), and 3 (D) were challenged 13 times with 20–30 TCID50 of SHIV-SF162P3 intravaginally (indicated by black arrows). Blood samples were drawn over 6 months and analyzed at indicated time points for plasma viral load determination. The horizontal black line indicates the lower detection limit of RNA copies/mL.

Gp41 Virosome Immunization Protects Female Monkeys from Repeated SHIV-SF162P3 Vaginal Challenges
One month after the last vaccination, all animals were challenged intravaginally thirteen separate times, biweekly for the first four weeks and then once a week for the remaining five weeks, with a low dose (20–30 TCID50) of the heterologous SHIV-SF162P3 (clade B, R5 tropism).
Viremia was investigated blindly for up to 208 days after the initial challenge (Figures 1B–1D). Starting after the ninth challenge, the six animals in control group 1 all became infected (Figure 1B), with viral loads peaking at 106 to107 copies/mL, as expected (Harouse et al., 2001). In group 2 (i.m. immunization), three of the animals were infected with peaks of viremia at 105 to 107 copies/mL, whereas two animals showed only transient peaks of viremia of ≤250 copies/mL and one consistently had no detectable viremia (Figure 1C). In contrast, only one of the animals in Group 3 (i.m.+i.n.) showed a low transient viral load of 800 copies/mL after the seventh challenge that returned to undetectable levels a week later, and all the other animals in the group had very low or undetectable amounts of viremia (Figure 1D).
For confirming the absence of infection in the viremia-negative animals, the presence of serum IgG against SIV p27gag antigen was investigated at day 100. As expected, the protected group 3 (i.m.+i.n.) animals and the viremia negative animals in group 2 (i.m. only) did not develop p27gag-specific serum IgGs. In contrast, all the infected animals from group 1 and group 2 showed a strong IgG response against p27gag SIV (Figure 2A). Furthermore, p27-specific IgAs were detected in the vaginal compartment of almost all viremia-positive animals, whereas the signal for specific mucosal IgG was very weak (Figure 2B). These results were confirmed with serum samples taken 3 months later (not shown).


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The absence of detectable p27-specific blood IgG and mucosal IgA in animals from groups 2 and 3 with undetectable viremia suggests that in these animals, local exposure to the p27 antigens during the 13 consecutive challenges with SHIV-SF162P3 was not sufficient to elicit the induction of gag-specific antibodies in detectable quantities. Furthermore, the fact that animal 3.3 from the i.m.+i.n.-vaccinated group did not seroconvert in spite of its transient viremia suggests that if infected, this animal rapidly resolved its infection.
Protection Is Not Associated with a Specific MHC Class I Haplotype
Specific MHC class I antigens (including Mamu-A*01, -A*02, -B*08, and -B*17) have been associated with SIV control (Loffredo et al., 2009). In the present study, all animals were negative for Mamu-A*01 and Mamu-A*02 (data not shown). There were two animals in group 2 (2.4 and 2.6) that were Mamu-B*08 positive, and one animal per group (1.1, 2.1, and 3.3) was positive for Mamu-B*17. These genetic traits did not apparently affect the results of the study.
Gp41 Virosome Vaccination Induces Both Plasma and Mucosal Antibody Responses
Because the virosomes used in the present study are not designed for triggering a CTL response (Amacker et al., 2005; Wilschut 2009), we limited our investigation to the humoral response. In the serum at week 24, the rgp41- and P1-specific IgG and IgA responses were observed in almost all animals in group 2 (i.m. only), whereas in group 3 (i.m.+i.n.), the response was not always detectable (Table 1). At the mucosal level at week 24, gp41- and P1-specific IgAs were detected in cervico-vaginal secretions (CVSs) and rectal secretions (RSs) of most immunized and protected animals. Regarding mucosal IgGs, only rgp41-specific IgGs, but not P1-specific IgGs, could be detected (not shown). A sizeable immune response required four immunizations as indicated by analyses of the samples at weeks 8 and 16 (Tables S1 and S2 available online). Overall, the mucosal immune response detected here may have been underestimated because detection of mucosal antibodies is always limited by the minute quantity of fluid that can be collected at each sampling time and by the dilution factor. Furthermore, the ELISA test used here only measured antibodies specific for linear and lipid-independent epitopes as we have shown earlier (Coutant et al., 2008).
Table 1. Evaluation of Antibodies toward the P1 and rgp41 Antigens in Each Animal
Sample
Serum
CVS
RS
Monkey Group 1:CT 2:i.m. 3:i.m.+i.n. 1:CT 2:i.m. 3:i.m.+i.n. 1:CT 2:i.m. 3:i.m.+i.n.
P1
IgA 0/6 5/6 4/5 0/6 5/6 5/5 0/6 5/6 2/5
IgG 0/6 6/6 3/5 0/6 6/6 2/5 0/6 0/6 0/5
rgp41
IgA 0/6 6/6 5/5 0/6 6/6 4/5 0/6 5/6 4/5
IgG 0/6 6/6 3/5 0/6 6/6 5/5 0/6 0/6 0/5
Systemic and mucosal responses to P1 and rgp41 in female Macaca mulatta monkeys after completion of the vaccination procedure with virosome-rgp41 and virosome-P1 (week 24 compared to week 0) in sera and CVS were analyzed by antigen-specific ELISA. For mucosal samples, results were calculated as the percent specific IgA or IgG of the total IgA or IgG in each sample, respectively, so as to account for the variation of total Ig between each mucosal sample. Samples showing a specific signal at least twice above the background were considered positive. Shown is the number of animals with specific IgAs or IgGs over the total number of animals in each group.

Taken together, these results indicate that the vaccine formulation was immunogenic, inducing both gp41-specific IgAs and IgGs in the systemic and mucosal compartments.
HIV-1-Blocking Antibodies Develop in the Mucosal Compartment, but Not in Serum
The functional inhibitory potential of the serum and mucosal antibodies in the vaccinated animals was investigated in an HIV-1 epithelial cell transcytosis assay ([Bomsel, 1997] and [Alfsen et al., 2001]). HIV-1-neutralizing activity was also studied with CD4+ T cells as target. The potential protective role of IgGs using not only their variable regions—that recognize the antigen—but also their Fc region was also evaluated in antibody-dependent cell cytotoxicity (ADCC) assay.
None of the sera of the vaccinated and protected animals at a dilution of 1/250 exhibited measurable neutralizing activity in any of the classical neutralization tests (data not shown), including inhibition of HIV-1 infection with TZM-bl cells and various recombinant viruses expressing primary envelopes, namely SF162, qh0692, and du172, SOS assays or CD4+ T cell assays with clade B R5-tropic HIV-1 JR-CSF (data not shown). Furthermore, ADCC mediated by serum could not be detected with P1 or recombinant gp160-coated CD4+ T cell targets (data not shown).
In contrast to serum, cervico-vaginal secretions (CVSs) exhibited various highly marked HIV-1 neutralization activities (Table 2, Figure 3). Table 2 shows that mucosal antibodies from CVS from the five animals in group 3 at a 6-fold dilution (corresponding to 0.2–15 μg of total antibodies/mL) could block HIV-1 transcytosis of a primary clade B, R5-tropic virus by 85%–100%. No blocking activity was detected in CVS samples from the placebo group. When tested against a primary clade C virus strain (Table 2), four out of five CVS samples could block transcytosis by more than 75%. These results demonstrate that the i.m.+i.n. immunization strategy could induce mucosal antibodies with in vitro cross-clade transcytosis-inhibition activity. Transcytosis-blocking activity in CVS samples from group 2 animals (i.m. only) was >90% for clade B and >60% for clade C viruses in two of the three uninfected animals but negligible in the infected animals. Pairwise group comparisons showed a significantly higher transcytosis-blocking activity in CVS from group 3, as compared to group 1 (placebo) for both clade B (p = 0.0054) (Figure 3A) and clade C (p = 0.0097) viruses (Figure 3B). Statistically significant differences, although lower, were also detectable between groups 2 (i.m.) and 1 (placebo) for both viral clades (p = 0.016 and 0.04, respectively).
Table 2. Transcytosis-Blocking, ADCC, and HIV-1 Neutralization Activities in Cervico-Vaginal Secretions, after the Last Immunization with P1- and rgp41-Virosomes
Transcytosis Inhibition (%)
ADCC Titer
Neutralization IC50 (CVS Dilution)
Monkey Clade B Clade C Specific for P1 Specific for gp41
Placebo
1.1 0 0 0 0 0
1.3 5 0 0 0 0
1.4 0 0 0 0 0
1.5 0 0 0 0 0
1.6 0 12 0 0 0
i.m.
2.1 28.2 65 27 9 6
2.2 3.1 4.5 3 3 6
2.3 2.0 0.75 3 3 6
2.4 89.8 12 3 0 6
2.5 96.0 71 27 3 0
i.m.+i.n.
3.1 96 99 27 9 6
3.3 94 73 3 3 0
3.4 96 85 3 0 6
3.5 100 42 27 9 0
3.6 85 78 27 9 18
Positive Controls
2F5 IgG 98 N.A. 800 pg/ml N.A. 15 μg/mL
98.6 IgG N.A. N.A. N.A. 32 pg/ml N.A.
For transcytosis blockade, preimmune (week 0) and immune (after the fourth immunization) (week 24) CVS at 1:6 dilutions from group 1 (placebo), group 2 (i.m.), and group 3 (i.m.+i.n.) animals were tested as described in Experimental Procedures and in the legend of Figure 3. For ADCC, preimmune and immune (after the fourth immunization) CVS at serial dilutions from 1:3 to 1:27 from groups 1, 2, and 3 were tested comparatively for P1- or rgp41-specific ADCC activity as indicated in Experimental Procedures. Results, obtained from at least two independent experiments, are expressed as ADCC titer as defined in Experimental Procedures. For neutralization, preimmune and immune (after the fourth immunization) CVS (1:6 dilution) from groups 1 and 3 were compared for their neutralizing activity against infection of CD4+ T cells with JR-CSF R5 tropic HIV-1 as indicated in Experimental Procedures. Results are expressed as the highest inhibitory CVS dilution resulting in 50% specific neutralization (IC50). Results were obtained from at least two independent experiments. Specific neutralization is defined by the neutralization observed in presence of immune CVS related to that observed in presence of preimmune CVS (week 24 % week 0).

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Figure 3.
HIV-1 Transcytosis-Blocking, ADCC, and Neutralization Activities in Cervico-Vaginal Secretions after Immunization with P1 and rgp41 Virosomes
For transcytosis-blockade, preimmune (week 0) and immune (after the fourth immunization: week 24) CVS at 1:6 dilution from group 1 (placebo), group 2 (i.m.), and group 3 (i.m.+i.n.) animals were preincubated with PBMCs infected with primary HIV-1 clade B 93BR029 (A) or clade C 92BR025 (B) prior to addition to the luminal side of polarized epithelial cell monolayers. Transcytosis inhibition is defined by the ratio of neutralization observed in presence of immune CVS related to that observed in presence of preimmune CVS (week 24 % week 0). Values are the mean (±SEM) of at least two independent experiments. For ADCC, preimmune and immune (after the fourth immunization: week 24) CVS at serial dilutions from 1:3 to 1:27 were tested comparatively for P1- (C) and rgp41- (D) specific ADCC activity as described in Experimental Procedures. Results are expressed as ADCC-specific lysis. Values are the mean (±SEM) of at least two independent experiments. For neutralization, preimmune and immune (after the fourth immunization: week 24) CVS (1:6 dilution) from groups 1, 2, and 3 were compared for their neutralizing activity against infection of human CD4+ T cells by JR-CSF R5 tropic HIV-1 as described in Experimental Procedures (E). Results are expressed as specific neutralization defined as the ratio of neutralization observed in presence of immune CVS related to that observed in presence of preimmune CVS (week 24 % week 0). Values are the mean (±SEM) of at least two independent experiments. For all functional tests, statistical analyses were performed first by the Kruskal-Walis test; pairwise comparisons were performed by the Mann-Whitney U-test, the p values are presented in each panel. KW statistic = 11.212, p = 0.0037 (A); KW statistic = 10.562, p = 0.0051 (B); KW statistic = 11.677, p = 0.0029 (C), KW statistic = 6.881, p = 0.0320 (D); KW statistic = 8.856, p = 0.0119 (E).

To determine which antibody isotype contained in CVS was actually involved in the prevention of HIV-1 transcytosis (Bomsel 1997), we depleted samples of IgA content and retested them. Figure 4 clearly demonstrates that no inhibition of transcytosis could be observed in the CVS samples depleted of IgAs. In contrast, IgG-depletion of the same samples had no effect on their transcytosis-blocking capacity. These results strongly suggest that the IgA fraction harbored the inhibitory activity, whereas the IgGs had no substantial role in preventing HIV-1 transcytosis in this model.


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Figure 4.
HIV-1 Transcytosis-Blocking Activities in Cervico-Vaginal Secretions from Group 3 after IgA or IgG Depletion
CVS from group 3 (white bars) were IgA- (black bars) or IgG- (gray bars) immuno-depleted prior to incubation with HIV-1-infected cells. After 2 hr contact with the mucosal barrier at 37°C, transcytosis was evaluated in the basal compartment by measuring the amount of translocated p24 gag protein and expressed as percent inhibition of transcytosis in presence of immune CVS (at week 24) related to percentage of transcytosis in the presence of preimmune CVS (at week 0). Error bars represent the means (±SEM) of at least two independent experiments.

As opposed to sera that contain very high, specific antibody concentrations upon vaccination, mucosal secretions generally contain a 100- to 1000-fold lower concentration of antibodies, which make their evaluation difficult in neutralization assays. CVS from group 3 generated limited neutralization activity for three out of the five monkeys when tested for neutralizing of human CD4+ T cell infection by HIV-1, a standard neutralization assay (Table 2, Figure 3E), although statistically significantly different from group 1 (placebo) (p = 0.0097, Figure 3E). In contrast, no biologically relevant neutralizing activity was found in CVS samples from groups 1 and 2.
Table 2 further shows that CVS IgGs from all group 3 animals exhibited substantial ADCC activity, regardless of the IgGs specificity for either P1 or rgp41, although P1-specific ADCC was always stronger. An ADCC activity was also detected for CVS IgGs from group 2 animals that were viremia negative, although with an overall lower ADCC titer. In contrast, IgGs from the placebo group (group 1) totally lacked ADCC activity. Pairwise group comparisons showed clearly a significantly higher P1-specific ADCC in CVS from group 3 (i.m.+i.n.) as compared to group 1 (placebo) (p = 0.0055) (Figure 3C), whereas the difference for rgp41-specific ADCC was less substantial (p = 0.028) (Figure 3D).
These sets of results were confirmed when comparing either viremia positive (including animals with low viremia blips fewer than 300 RNA copies/ml) versus viremia negative animals (Figures S1A–S1D) or when comparing persistently infected versus protected animals (Figures S2A–S2D).
Finally, for all animals taken together, a strong highly significant inverse correlation was observed between clade B and clade C HIV-1 transcytosis-blocking activity in CVS and peak acute viremia (Figures 5A and 5B) (r = −0.832; p < r =" −0.850;" r =" −0.682;" p =" 0.0036)">80% the in vitro transcytosis of primary HIV-1 viruses from clades B and C, suggesting cross-clade recognition. This is particularly interesting, given that the tested HIV-1 clade C virus does not have the 2F5 and 4E10 motifs in its gp41 sequence. This suggests that other functional epitopes and/or 3D epitope(s) might be shared between the clade B and C gp41 molecules, despite the absence of linear epitope identity. HIV-1-blocking IgAs specific for such cross-clade epitopes in HIV-1 envelope (Devito et al., 2002) and more specifically in P1 are detected in HEPS individuals (Tudor et al., 2009) and were also induced in monkeys upon intramuscular or mucosal immunization with virosome-P1 alone.
Furthermore, the transcytosis-blocking activity observed here at the mucosal level in the CVS of the i.m.+i.n.-vaccinated animals relied almost entirely on IgAs because IgA depletion, but not IgG ones, totally abolished transcytosis blockade in vitro. These findings are in agreement with previous studies showing that gp41-dimeric IgAs and secretory IgAs could either block HIV-1 intracellularly during transcytosis and redirect it to the mucosal surface (Bomsel et al., 1998) or impair its mucosal entry ([Devito et al., 2000a], [Alfsen et al., 2001], [Broliden et al., 2001] and [Miyazawa et al., 2009]). IgAs have also been shown to retro-translocate pathogens from the lamina propria into the lumen (Corthésy 2007).
Taken together, our results strongly suggest that vaccine-induced mucosal gp41-specific IgAs efficiently prevented HIV-1 binding to the mucosal receptor galactosyl ceramide (Alfsen and Bomsel, 2002) or to other gp41 epitopes such as the caveolin binding motif (Hovanessian et al., 2004), thus blocking initial virus entry in mucosal tissues. This conclusion will have to be strengthened and confirmed in further studies.
IgGs constitute an important fraction of vaginal antibodies, whereas in other mucosal secretions IgAs predominate. P1- and/or rgp41-specific ADCC activity mediated by mucosal IgGs could also be detected in protected animals from group 3 and animals from group 2 with undetectable or transient viremia ≤250 viral RNA copies/mL. In contrast, CVS from the infected animals in group 2 showed low IgG-dependent ADCC activity and lacked transcytosis-blocking activity. Therefore, it is possible that transcytosis inhibition must be combined with ADCC to block the initiation of HIV-1 infection at mucosal sites. Indeed, previous studies have associated mucosal ADCC activity with lower blood viremia in HIV-1-positive women (Nag et al., 2004), and disappearance of ADCC seems to correlate with lower CD4+ T cell counts and progression to AIDS in men and in the nonhuman primate model (Xiao et al., 2010 and reviewed in Forthal and Moog, 2009). The discrepancy between serum and mucosal IgG activities confirms the presence of locally produced IgGs ([Ogra and Ogra, 1973], [Brandtzaeg, 2009] and [Bouvet et al., 2002]) and the compartmentalization of the mucosal immune response ([Bouvet and Fischetti, 1999] and [Macpherson et al., 2008]).
Knowing that the mucosal immune response is relatively short lived, compared with blood antibodies, it will be important to determine in future studies the duration of the protective immune response observed here.
The most remarkable finding in this study was that mucosal protection against a heterologous SHIV challenge in rhesus macaques occurred in the absence of detectable neutralizing antibodies in the serum. This is reminiscent of data on the protection induced by injection of a panel of HIV-1-envelope-specific IgGs (Hessell et al., 2009; 2010) and clearly challenges the paradigm that mucosal protection requires the presence in serum of high titers of IgGs with virus-neutralizing capacity.
Altogether, this study demonstrates that a vaccine that stimulates the production of HIV-1 envelope gp41-specific antibody in the vaginal tissue was sufficient to protect monkey from vaginal exposure to virulent virus. This may serve toward the development of a human vaccine against HIV-1 and acquired immunodeficiency syndrome and help to explain why a few individuals who lack HIV-1-specific antibodies in the blood are able to resist infection, even when they are repeatedly exposed to HIV-1.
Experimental Procedures
Antigens
The P1 sequence (649-683) (originally described in Alfsen and Bomsel, 2002) derived from the HXB2 clade B HIV-1 strain and was modified to a lipid-linked synthetic peptide having 38 amino acids with the following sequence, SQTQQEKNEQELLELDKWASLWNWFDITNWLWYIKLSC, linked to phosphatidyl-ethanolamine (PE) via its C-terminal cysteine. The rgp41 protein was derived from the amino acid sequence 540-663 of HXB2 clade B HIV-1 strain and was produced in E. coli with the following sequence, MQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLSGGRGGSSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQLLELLEHHHHC, (italic letters correspond to the overlap region with the P1 peptide). The molecule was covalently linked via a bifunctional succinate linker to PE. This rgp41 is lacking the binding motifs for the mucosal receptor galactosyl ceramide (Alfsen and Bomsel, 2002) and the 2F5 and 4E10 motifs ([Muster et al., 1993] and [Stiegler et al., 2001]). A deletion of 25 amino acids (593–618) was introduced into the molecule to keep it soluble and trimeric, as well as point mutations to greatly reduce 3D homology with the human IL-2, as previously published (Delcroix-Genête et al., 2006). The rgp41 was purified through several steps of chromatography as described (Delcroix-Genête et al., 2006), resulting in a stable trimeric protein of 39.9 kDa.
Virosomes
Influenza virus strain A/Singapore/6/86 (H1N1) was cultivated in embryonated hen eggs and inactivated with β-propiolactone before solubilization with 100 mM octaethylene glycol monododecyl ether in phosphate-buffered saline-NaCl (OEG-PBS). The solubilized viral hemagglutinin (HA) and neuramidase (NA) were mixed with egg phosphatidylcholine (PC) and synthetic phosphatidylethanolamine and the antigens of interest, P1 or rgp41, previously conjugated to PE. Detergent was removed on polystyrene beads on two subsequent batch chromatography steps, and homogenous influenza virosomes, that spontaneously reconstituted, were filtered for sterilization without further purification (Amacker et al., 2005) (Kammer et al., 2007).
Immunization
Four weeks prior to vaccination, female Macaca mulatta monkeys of Chinese origin were preimmunized with inactivated influenza (A/Singapore/6/86) strain, propagated in the allantoic cavity of embryonated eggs obtained from Berna Biotech AG and purified as described previously (Amacker et al., 2005) to mimic the situation in human usually immune to influenza. Monkeys were then subdivided into three groups of six animals each. Each animal received four vaccine injections, at weeks 0, 7, 15, and 23. The placebo control group (group 1) received intramuscular injections of IRIV (virosome alone; no HIV-1 antigen). Groups 2 and 3 received vaccine doses containing each 40 μg of P1 peptide and 40 μg of rgp41. Group 2 received only intramuscular (i.m.) injections, whereas group 3 received two i.m. injections at weeks 0 and 7 and then two intranasal (i.n.) injections at weeks 15 and 23. For intramuscular injections, each dose (100 μl volume) was administered in one site (upper leg), whereas intranasal administrations were equally distributed between the two nostrils (50 μl per nostril) with a specific spray device (BD Medical Devices).
Animals were maintained under guidelines established by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals, with protocols approved by the local ethical committee.
Samples Harvesting
Blood, vaginal, and rectal washes were performed on each animal at week −4 and 0 for preimmune samples and 1 week after each vaccination event for immune samples (weeks 1, 8, 16, and 24). Cervico-vaginal and rectal secretions were obtained by gentle lavages at indicated times with 3 ml of cold saline buffer, then added with antibiotics and protease inhibitors and centrifuged. Resulting supernatants were immediately aliquoted and snap frozen in liquid nitrogen before storing at −80°C (Tudor et al., 2009).
ELISA
Total antibodies were measured by standard sandwich ELISA, whereas P1- or rgp41-specific antibodies were evaluated by P1- or rgp41-coated direct ELISA ([Coutant et al., 2008] and [Tudor et al., 2009]) with monkey specific-IgAs or IgGs coupled to HRP (Rockland) as secondary antibodies.
Challenges
SHIV-SF162P3 (R5 clade B) was kindly provided by N. Miller, NIAID (NIH AIDS Research and reference reagent program). The genome of this chimeric simian-human immunodeficiency virus contains the env (gp120 + gp41), tat, rev, and vpu genes from HIV-1 SF162 (Harouse et al., 2001) inserted into the genome of the pathogenic SIVmac239. One month after vaccination, all animals were challenged intravaginally 13 times with 2 ml of a phosphate-buffer viral solution containing 20 TCID50 of the heterologous SHIV-SF162P3 for the first 7 challenges and 30 TCID50 for the 6 last challenges. Challenges were done every 4–7 days.
Viremia
Blood samples were drawn over 6 months, twice a week for the first 2 weeks, once a week for the subsequent weeks, and then once every 2 weeks, and analyzed for plasma viral loads (viremia) with the QIAGEN QuantiTect SYBRGreen RT-PCR kit with specific SIV gag probes (sensitivity 1000 copies/mL blood), then repeated blindly as described (Li et al., 2009) with a more sensitive technique. In brief, viral RNA from 0.2 ml of EDTA-anti-coagulated cell-free plasma was directly extracted by a MagNa Pure LC robotic workstation (Roche Molecular Biochemicals). A one-step reverse transcriptase PCR (RT-PCR) method using the TaqMan EZ RT-PCR CORE REAGENT kit was performed for quantification of SHIV viral RNA. Standard curves were prepared with a series of six 10-fold dilutions of viral RNA of known concentration. The sensitivity of the assay theoretically was 100 RNA equivalents per mL, but because of volume issues, it was fixed at 200 copies/mL. Samples were analyzed in triplicate and the number of RNA equivalents was calculated per milliliter of plasma.
P27 Antibody Detection
Western blots with serum (diluted 1/100 for IgA, 1/200 for IgG) and cervico-vaginal secretions (diluted 1/6) were performed in accordance with the manufacturer's instruction (New Lav Blot I from Bio Rad) as described (Tudor et al., 2009) but with a monkey-specific secondary antibody coupled to HRP (Rockland).
Functional In Vitro Assays
Inhibition of HIV-1 transcytosis ([Alfsen et al., 2001] and [Tudor et al., 2009]) and TZM-bl (Pastori et al., 2008) (Bomsel et al., 2007), SOS (Pastori et al., 2008) (Bomsel et al., 2007), and CD4+ T cell neutralization assays ([Alfsen et al., 2001] and [Tudor et al., 2009]) were performed as previously described. For the CD4+ T cell neutralization assays using the JR-CSF, R5 tropic HIV-1 molecular clone, specific neutralization was expressed as follows:
Specific neutralization = 100 - 100 X (Infection in presence of secretion collected at week 24/Infection in presence of secretion collected at week 0)
For the transcytosis assays, clade B (93BR029) and C (92BR025) HIV-1-infected PBMCs were used to inoculated HEC-1 endometrial cell lines cultured in a polarized manner in a two-chamber system as described ([Alfsen et al., 2001] and [Tudor et al., 2009]). CVS were tested at dilutions 1:6 and 1:12 and the observed transcytosis-blockade was directly dependent on CVS concentration (not shown). 2F5 IgA (1 μg/mL) (Shen et al., 2010) was used as positive control and inhibited transcytosis by 95%.
Where indicated, cervico-vaginal secretions were depleted of IgAs or IgGs by immunodepletion. In brief, biotinylated-anti-human IgA or biotinylated-anti-human IgG (Caltag) were bound to Streptavidin-Agarose (Pierce), 10 μg/30 μl of beads. Beads coupled with anti-human IgA or IgG antibody were washed 3 times to removed unbound biotinylated anti-IgA or anti-IgG, after which 30 μl of anti-IgA- or anti-IgG-bound beads were incubated overnight at 4°C on a rotating wheel with CVS (1/6 dilution). The mixture was centrifuged for 10 min at 10 000 g at 4°C and the resulting supernatant represented the IgA- or IgG-depleted CVS fraction. Transcytosis activity is expressed as follows: transcytosis inhibition = 100 – 100 X (transcytosis in presence of secretion collected at week 24/ transcytosis in presence of secretion collected at week 0).
For the TZM-bl assays (Tudor et al., 2009), three different HIV-1 strains were tested: SF162, the primary clade B QH0692, and the primary clade C DU172. Virus neutralization was evaluated with the following positive controls: 2F5 IgG, which gave a 50% inhibitory concentration (IC50) of 3 μg/mL (range: 5–2 μg/mL), and an IC90 of 50 μg/mL (range 67–34 μg/mL); the TRIMAB gave an IC50 of 0.9 μg/mL (range: 0.7–1.7 μg/mL) and an IC90 of 5 μg/mL (range: 4–6 μg/mL).
For the SOS assays (Bomsel et al., 2007) evaluated on U87.CCR5.CD4 target cells, the positive controls were 2F5 IgG with an IC50 of 2.2 μg/mL (range 3.5–1.7 μg/mL) and IC90 of 21 μg/mL (range: 18–32 μg/mL).
Of note, SF162 and JR-CSF envelopes have 88% sequence homology, with the differences located in the variable sequences of gp120 but not in gp41; therefore, they are very similar in terms of gp41 sequence.
ADCC assay was performed as described (Xiao et al., 2010), with THP1 monocytic cells as effector cells and CCR5+ -NK resistant CEM lymphoid cells (NIH AIDS reagents program) coated with either P1 or recombinant X4 tropic gp160 as target cells. In brief, 1 × 106 CEM-NKR-CCR5 target cells were incubated with 0.2 μM of gp160 (MN/LAI Pateur-Merieux) or 5 μM of P1 peptide for 1 hr at room temperature in a total volume of 300 μl of RPMI. Coated cells were dually labeled with the membrane dye PKH-26 (Sigma-Aldrich) and the vital dye CFSE (Molecular Probes, Invitrogen), then incubated for 30 min at room temperature with the samples (3-fold dilutions) or the control antibodies, 2F5 IgG for P1-specific ADCC and 98.6 IgG for rgp41-specific ADCC, respectively, in a 96-well microtiter plate. Effector THP1 cells were then added at a relatively low effector/target ratio of 10:1 (Xiao et al., 2010). The reaction mixture was incubated for 4 hr at 37°C in 5% CO2 after which the cells were immediately analyzed with a Becton Dickinson FACSCalibur flow cytometer. Data analysis was performed with Cytomix RXP software. We determined the percentage of ADCC cell killing by back-gating on the PKH-26high population of target cells that lost the CFSE viability dye. For each gp41 subunit, a specific ADCC was expressed as percent ADCC in the presence of sample collected at week 24 subtracted by the percentage of ADCC in the presence of preimmune sample. ADCC titers were defined as the reciprocal dilution at which the percent ADCC killing was greater than the mean percent killing of the negative controls plus three standard deviations. Data represent the mean of at least two independent experiments. The positive control for P1-specific ADCC, 2F5 IgG at 1 μg/mL resulted in 40% specific cell lysis, and that for rgp41-specific ADCC, 98.6 IgG at 100 ng/mL resulted in 60% specific cell lysis. Both signals were entirely reversed by a 10-fold excess of non-specific IgG (not shown), indicating that the ADCC measured was IgG-dependent. Furthermore, effector cells did not bind IgA or expressed the CD89 Fcα receptor (not shown).
Antibody Purification
Antibodies contained in preimmune and immune cervico-vaginal secretions were purified on a protein G (IgG) or anion-exchange (IgA) column with an automatic HPLC system. Eluted fractions in PBS were evaluated by ELISA using goat anti-human IgG, IgA, or IgM as described (Donadoni et al., 2010).
MHC Genotyping
It was performed by sequence-specific PCR as described (Loffredo et al., 2009).
Statistical Analysis
For comparing functional activities of cervico-vaginal secretions among monkey groups, the nonparametric Kruskal-Wallis test was used. Pairwise comparisons were performed by the nonparametric Mann-Whitney U test. The Spearman rank correlation test was used for assessing the relationship between viral load and transcytosis-blockade, ADCC, and neutralization, respectively. Statistical analyses were carried out with Instat GraphPad Software, version 5.0.
Acknowledgments
We are grateful to C. Pastori (San Raffaele Scientific Institute, Milano, Italy) for technical help, to B. Weksler (Cornell University, New York, USA) for English editing, to Pr M. Girard for fruitful discussions and review of the manuscript, and to C. Rochet (Mymetics, Switzerland) for constant encouragement and support during our collaboration. L.L. and M.B. were supported by grant no. 201433 from European Commission/Seventh Framework Programme (www.ngin.eu) and L.L. by a grant (no. 1 U19 AI062150) from NIH and grants GCE no. 53030 and no. PP1008144 from Bill and Melinda Gates Foundation. M.B. was supported by grants from Agence Nationale de Recherche sur le SIDA et les Hépatites (ANRS), SIDACTION-Ensemble contre le SIDA, and La Fondation pour la Recherche Médicale (FRM). Y.G. was supported by SIDACTION and ANRS. R.Z. was Chief Scientific Officer and R.Z. and M.A. are shareholders of Pevion Biotech Ltd., which provided the vaccine. S.F. is Chief Scientific Officer and a member of the Executive Board of Mymetics Corp., A.C. is Scientific Officer of Mymetics Corp. G.D. is employee of BD Medical-Pharmaceutical, and M.G.R. and N.M. are employees of PX’ Therapeutics .
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