There were 94 deaths in each treatment group (hazard ratio 0.97, 95% CI 0.73–1.30, P = 0.86). The causes of death as reported by relatives or confidantes did not differ between the two treatment groups, including deaths attributed to TB, AIDS, and other febrile illnesses. The proportion of patients with one or more serious adverse events was similar in both groups (Table 4); events attributed to malaria (including some clinical diagnoses and some false-positive smears, thus some ‘malaria’ cases were possibly due to other causes of fever such as TB) were significantly lower in M. vaccae recipients. No adverse effects were noted on CD4 cell count or HIV viral load (data not shown). Repeat TST data on 215 patients showed that among those with a TST of less than 10 mm at baseline, the frequency of a TST of at least 10 mm following the fifth dose was similar in both treatment groups [8/81 (9.9%) M. vaccae vs. 8/71 (11.3%) placebo; P = 0.92]. No patients were observed to have a Koch reaction.
We chose disseminated or bacteremic TB as the primary study endpoint based on our prior studies [24,42–44], indicating that this form of disease is common in advanced HIV infection (CD4 cell count <75 cells/μl). Further, it is strictly defined, more readily classified than pulmonary TB, and might represent the most sensitive indicator of a biologic effect of immunization (e.g., BCG is most effective in preventing miliary and meningeal TB, which are likely mediated via bacteremia ). At the time of termination, only 20 cases of disseminated TB had been detected compared with the 71 expected cases. Factors that may have directly reduced the rate of disseminated TB include prevention of dissemination of TB by aggressive surveillance for and treatment of the earlier complication of pulmonary TB, the increasing use of ART over time, administration of INH to patients with positive TSTs, and the return of patients with advanced AIDS to their home villages, making follow-up less complete for this endpoint. The observed reduction in disseminated TB cases (seven M. vaccae vs. 13 placebo) paralleled the statistically significant reduction in definite TB cases.
Definite TB, defined here as a secondary endpoint, is the usual primary endpoint in trials for the prevention of TB [3,45], and protection was significant against this rigorously defined and clinically meaningful endpoint. The lack of M. vaccae-induced protection against the other secondary endpoint, clinically defined or ‘probable’ TB, is parallel to the finding in the recent update by Aronson et al.  of the 3287 persons BCG trial conducted in the western United States in the 1930s. BCG was found to offer significant protection against culture-confirmed TB for as long as 50–60 years but no protection against clinical/probable TB (nine probable TB cases in BCG recipients and three probable cases in the placebo group) . In the one, other successful BCG trial from the meta-analysis by Colditz et al.  of prospective trials, which included definitions for both culture-confirmed definite TB and clinically categorized probable TB, vaccine efficacy was lower for probable than definite TB . There are two possible explanations for this repeated observation in TB vaccine trials: case misclassification or a fundamental and as yet unidentified characteristic of ‘clinical’ or probable TB. In either case, future trials of new vaccines against TB should carefully distinguish definite vs. probable cases of TB.
These data conclusively establish the safety of this inactivated whole-cell mycobacterial vaccine in persons with HIV and are consistent with those reported in other trials of M. vaccae in persons without HIV [30,31]. However, the major differences in both the regimens and objectives for our M. vaccae trial compared with previous ones are important to note. We examined the ability of multiple-dose M. vaccae to boost immunity and prevent TB in persons primed by BCG. Previous trials studied single-dose M. vaccae as an adjunct to treatment of drug-susceptible TB [30,31]. Inactivated whole-cell vaccines must be given in a multiple-dose schedule to obtain an optimal preventive immune response .
Although morbidity due to TB was reduced by immunization, we did not observe an effect on mortality. In our research setting, all patients were followed closely, had TB diagnosed promptly, sometimes before the onset of clinical symptoms or radiologic abnormalities [52,53], and were referred for ART. It is possible that a mortality benefit might accrue among patients with more limited access to medical care, with delayed diagnosis and treatment of TB. Only a limited number of patients in this trial were receiving ART. Although there was a trend toward lower vaccine efficacy in those receiving ART, the difference was not statistically significant.
Notable strengths of the trial include that it was randomized, placebo-controlled, double-blinded, and well executed. We were able to achieve high rates of compliance with a five-dose schedule, a regimen that might encourage closer follow-up of patients with HIV infection. However, we have shown that a three-dose schedule is immunogenic , and further study of this more practical schedule should also be considered.
We thank Drs Neal Halsey, Clyde Crumpacker, Kenneth McIntosh, Andrew Swai, Paige Williams, Mary Wilson (members of the DSMB); Drs Paul Fine, Daniel Hoft, Marja-Leena Katila, John F. Modlin, Christopher Whalen (consultants), Dr Barry Kreiswirth (molecular typing), Nancy Wray (administrative support), Louise Grant (MB/BacT, bioMerieux), David Kolesar (Isolator, Inverness), Laura Rose Brunet (Silence Therapeutics), Betty Mchaki, Outi Rautio, other laboratory and clinical staff and the many Tanzanian participants who volunteered for the study. We thank Dr Jerald Sadoff of the Aeras Global Tuberculosis Vaccine Foundation for helpful comments.
This study is supported by grants from the NIH, DAIDS, AI 45407 and the Fogarty International Center, D43-TW006807. Vaccine for the study was provided by SR Pharma (London). The study is registered at ClinicalTrials.gov as NCT00052195.
There are no conflicts of interest reported. Dr Vuola is now with GlaxoSmithKline, Europe.
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HIV viral load was performed at baseline until August 2003. Blood was obtained at baseline and after dose 5 for immunologic assays including lymphocyte proliferation assays and interferon gamma release assays in response to four mycobacterial antigens (these studies are the subject of a separate report). Persons with tuberculin skin test reactions (TST) ≥ 5 mm at baseline and no history of prior treatment for tuberculosis were given isoniazid 300 mg daily for 6 months and followed monthly during this interval . TSTs were repeated 2 months after dose 5.
Subjects were randomized 1: 1 in blocks of 6–14 to receive a 5-dose series of 0.1 mL intradermal Mycobacterium vaccae (MV; SRL 172; batch MV 017 until July 31, 2004 then batch S00631; Silence Therapeutics, London) or placebo (P; borate buffered saline in an identical vial) on the forearm at 0, 2, 4, 6, and 12 months.
In Substudy A (safety), the first 165 subjects were scheduled to return at one and four weeks after each dose of vaccine for detailed examination of vaccine site reactions and for interviews on local and systemic side effects (Table A). Subjects also had HIV viral load and CD4 count determined 2 months after each dose of vaccine. In Substudy B (immunogenicity), the next 150 subjects had vaccine immunology performed 2 months after dose 3 and 5, and annually. Subjects in both Substudy A and Substudy B had repeat tuberculin skin testing performed 2 months after dose 3 and 2 months after dose 5.
Patients with suspect tuberculosis were referred to the National Tuberculosis and Leprosy Control Program (NTLP) with results of these evaluations for decisions about treatment. Standard tuberculosis treatment at the inception of the study was directly observed isoniazid, rifampin, ethambutol and pyrazinamide for 2 months followed by isoniazid and ethambutol for another 6 months. On February 1, 2006 NTLP treatment guidelines were changed to directly observed isoniazid, rifampin, ethambutol and pyrazinamide for 2 months followed by isoniazid and rifampin for another 4 months. Follow up data on treatment outcome were obtained by interview and examination of patients at 2, 5 and 8 months of treatment (and after the change in treatment policy in 2006 at 2, 4 and 6 months).
All subjects were treated for intercurrent infections at the study site or by referral to district hospitals for inpatient care. At each visit, subjects were evaluated to determine if they met current national criteria for initiation of antiretroviral therapy (ART). ART-eligible subjects were counseled and referred to a Tanzanian Ministry of Health Care and Treatment Center for ART. Dates and putative causes of death were obtained from interviews with a relative or confidante named at the time of study entry, and, less commonly, from treating medical facilities and personnel.
Sputum AFB stains were performed using the Ziehl-Neelsen method. Mycobacterial sputum cultures were performed at the study laboratory in Tanzania using standard methods: 1% NaOH decontamination followed by culture on Lowenstein-Jensen agar slants with 10 weeks of air incubation. Blood cultures were processed by inoculation of 5 mL blood into an automated broth system with a 6 week incubation period (MB/BacT, bio-Merieux, Lyon, France) with back-up use of the Isolator mycobacterial blood system (Inverness Medical Professional Diagnostics, Princeton, NJ) at times when the automated system was not operational . Beginning November 19, 2004 a 6 week terminal subculture to Lowenstein-Jensen medium was performed on all MB/BacT negative blood cultures. AFB positive isolates were shipped to Dartmouth Medical School and tested for the M. tuberculosis complex (MTBC) using DNA probes (Accuprobe, Gen-Probe, San Diego, CA).
IS6110 typing was performed on study isolates and concurrent community isolates obtained from the NTLP laboratory by Dr. Barry Kreiswirth using standard methods . Isolates with IS6110 patterns that were identical or differed by 1 band were examined for date of processing to determine possible cross contamination . The date processed (or date collected if former was unavailable) was reviewed for matching patterns to identify clusters that may have represented false positive results due to either laboratory cross contamination or mislabeling. Date parameters used to define clusters were ±4 days from date processed and ±10 days from date of specimen collection.
CD4 counts were performed by flow-cytometry (FACSCount, Becton Dickinson, Franklin Lakes, New Jersey). Viral loads were performed using Versant HIV-1 RNA 3.0—bDNA (Bayer Diagnostics, Emeryville, CA) or Versant 340 bDNA (Siemens Diagnostics, Norwood, MA).
The trial was designed to detect with 80% power a 50% decrease in the risk of disseminated tuberculosis at the 0.05 (2-sided) statistical significance level. An annual disseminated tuberculosis rate of 1.60% and an annual death rate of 9.16% were expected in the placebo group. Follow-up loss was expected to be 27% over the entire study duration. Based on these assumptions, a total of 71 events was needed and was expected to be observed among 2274 participants followed over a median of four years.
One interim efficacy analysis for the primary endpoint of disseminated tuberculosis was conducted in November 2005 when 9 cases had been observed. This resulted in an information time of 0.13 with a corresponding critical value using an O-Brien Fleming boundary of 6.206. The DSMB reviewed the log rank tests and compared their P values to the nominal P value at the interim analysis, which did not indicate a significant difference warranting stopping (or considering to stop) the trial.
Among 246 ineligibles whose reason was ‘other’ (consort diagram) subcategories were: failure to return – 79, HIV negative – 43, no CD4 count – 52, serious underlying disease – 26, refused to continue – 18, moved – 8, screening incomplete when enrollment stopped – 20. Subjects who were lost to follow-up compared to subjects who remained in the study were less often female (69% vs. 77%, respectively, P < 0.0001), less likely to have had prior tuberculosis (4.4% vs. 9.3%, P < 0.001), and less likely to have a positive TST (27% vs. 33%, P = 0.02); age, sex, and baseline CD4 counts did not differ between the two groups.
The following number of subjects received the following minimum number of doses: one dose 1975 (988 MV, 987 P); two doses 1902 (950,952); three doses 1853 (928, 925); four doses 1804 (903, 901); five doses 1687 (848, 839).
The HR for definite plus probable tuberculosis in the ITT analysis was 0.83 (95% CI: 0.61–1.13; 79 MV, 92 P; P = 0.22) and in the PP analysis was 0.86 (95% CI: 0.63–1.17; 78 MV, 90 P, P = 0.31). Two MV subjects had episodes of both definite and probable tuberculosis; only one episode was included for each of these subjects in the definite plus probable calculation.
M. tuberculosis was isolated from 81 subjects; 71 study isolates and 168 concurrent community control isolates were available for IS6110 typing. Five clusters (four with identical IS6110 results and one with similar IS6110 results) were identified and reviewed by the expert panel. One cluster was determined to represent a false positive result and resulted in the change of one tuberculosis case classification from definite to unlikely.
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