Sustained improvements in handwashing indicators more than 5 years after a cluster-randomised, community-based trial of handwashing promotion in Karachi, Pakistan.

OBJECTIVE: To evaluate handwashing behaviour 5 years after a handwashing intervention in Karachi, Pakistan. METHODS: In 2003, we randomised neighbourhoods to control, handwashing promotion, or handwashing promotion and water treatment. Intervention households were given soap +/- water treatment product and weekly handwashing education for 9 months. In 2009, we re-enrolled 461 households from the three study groups: control (160), handwashing (141), and handwashing + water treatment (160) and assessed hygiene-related outcomes, accounting for clustering. RESULTS: Intervention households were 3.4 times more likely than controls to have soap at their handwashing stations during the study visit [293/301 (97%) vs. 45/159 (28%), P < 0.0001]. While nearly all households reported handwashing after toileting, intervention households more commonly reported handwashing before cooking [relative risk (RR) 1.2 (95% confidence interval (CI) 1.0-1.4)] and before meals [RR 1.7 (95% CI, 1.3-2.1)]. Control households cited a mean of 3.87 occasions for washing hands; handwashing households, 4.74 occasions; and handwashing + water treatment households, 4.78 occasions (P < 0.0001). Households reported purchasing a mean of 0.65 (control), 0.91 (handwashing) and 1.1 (handwashing + water treatment) bars of soap/person/month (P < 0.0001). CONCLUSIONS: Five years after receiving handwashing promotion, intervention households were more likely to have soap at the household handwashing station, know key times to wash hands and report purchasing more soap than controls, suggesting habituation of improved handwashing practices in this population. Intensive handwashing promotion may be an effective strategy for habituating hygiene behaviours and improving health.

Association between intensive handwashing promotion and child development in Karachi, Pakistan: a cluster randomized controlled trial.

OBJECTIVE: To evaluate associations between handwashing promotion and child growth and development. DESIGN: Cluster randomized controlled trial. SETTING: Informal settlements in Karachi, Pakistan. PARTICIPANTS: A total of 461 children who were enrolled in a trial of household-level handwashing promotion in 2003 and were younger than 8 years at reassessment in 2009. INTERVENTIONS: In 2003, neighborhoods were randomized to control (n = 9), handwashing promotion (n = 9), or handwashing promotion and drinking water treatment (n = 10); intervention households received free soap and weekly handwashing promotion for 9 months. MAIN OUTCOME MEASURES: Anthropometrics and developmental quotients measured with the Battelle Developmental Inventory II at 5 to 7 years of age. RESULTS: Overall, 24.9% (95% CI, 20.0%-30.6%) and 22.1% (95% CI, 18.0%-26.8%) of children had z scores that were more than 2 SDs below the expected z scores for height and body mass index for age, respectively; anthropometrics did not differ significantly across study groups. Global developmental quotients averaged 104.4 (95% CI, 101.9-107.0) among intervention children and 98.3 (95% CI, 93.1-103.4) among control children (P = .04). Differences of similar magnitude were measured across adaptive, personal-social, communication, cognitive, and motor domains. CONCLUSIONS: Although growth was similar across groups, children randomized to the handwashing promotion during their first 30 months of age attained global developmental quotients 0.4 SDs greater than those of control children at 5 to 7 years of age. These gains are comparable to those of at-risk children enrolled in publicly funded preschools in the United States and suggest that handwashing promotion could improve child well-being and societal productivity. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01538953.

Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

Effect of alternative aluminum adjuvants on the absorption and immunogenicity of HPV16 L1 VLPs in mice.

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO(4)), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16. Electron microscopy demonstrated that the physical appearance of the phosphate-containing aluminum adjuvants was markedly different from that of aluminum hydroxide. All three aluminum adjuvants were found to display unique surface charge profiles over a range of pH, while AAHS demonstrated the greatest inherent capacity for adsorption of L1 VLPs. These differences were associated with differences in immunogenicity: anti-HPV L1 VLP responses from mice immunized with AAHS-formulated HPV16 vaccine were substantially greater than those produced by mice immunized with the same antigen formulated with aluminum hydroxide. In addition, HPV L1 VLPs formulated on AAHS also induced a substantial interferon-gamma secreting T cell response to L1 peptides indicating the potential for an enhanced memory response to this antigen. These results indicate that the chemical composition of aluminum adjuvants can have a profound influence on the magnitude and quality of the immune response to HPV VLP vaccines.

Enhanced rates and magnitude of immune responses detected against an HIV vaccine: effect of using an optimized process for isolating PBMC.

Quantitative analysis of cell-mediated immune responses induced by candidate HIV vaccines requires robust procedures for collecting and processing human peripheral mononuclear blood cells (PBMCs). We evaluated several parameters in order to optimize a sample handling process that would be suitable for a multicenter clinical trial. Among the findings, systematic increases in the magnitude of IFN-gamma ELISpot responses were observed when the time from blood collection to PBMC freezing was reduced to <12 h. By implementing these improvements within an ongoing clinical trial, the estimated immunologic response rates to an adenovirus- based HIV vaccine increased by more than 20 percentage points to approximately 80% of the vaccine recipients against any of the vaccine antigens and the average levels of T cell response improved more than 3-fold. These studies establish the importance of optimal conditions for PBMC collection and handling to the success of a clinical development program.

Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay.

An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.

A comparison of standard immunogenicity assays for monitoring HIV type 1 gag-specific T cell responses in Ad5 HIV Type 1 gag vaccinated human subjects.

Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.

Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag.

The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(-) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(-) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Identification of T-cell epitopes using ELISpot and peptide pool arrays.

Here we describe a method for T-cell epitope identification using a modified ELISpot assay that is both simple and efficient. By using a carefully constructed array of pools of overlapping peptides spanning the entire antigen sequence to stimulate T-cell responses, we are able to detect antigen-specific cytokine responses by both CD8+ and CD4+ T cells and identify the specific peptides to which the cells are responding. Additionally, by performing magnetic bead depletion of either CD8+ or CD4+ cells prior to the assay, we are able to determine the phenotype of the responding cells to each of the peptide epitopes identified. Use of this method will allow the identification of both CD4+ and CD8+ T-cell epitopes without the need for MHC allele-matched reagents and without the need for highly specialized instrumentation. By using an array of peptide pools, this method also dramatically reduces the number of immune cells required to test the entire antigen sequence, often a limiting factor in vaccine testing and other studies.

Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models.

Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.

Effect of vaccine delivery system on the induction of HPV16L1-specific humoral and cell-mediated immune responses in immunized rhesus macaques.

There have been numerous studies to assess the immunogenicity of candidate therapeutic and prophylactic vaccines for human papillomavirus (HPV), but few of them have directly compared different vaccines in an immunologically relevant animal system. In the present study, several vaccine delivery systems (VLPs, chimeric VLPs, plasmid DNA, and a replication incompetent adenoviral vector) expressing HPV16L1 were evaluated for their ability to induce HPV16L1 VLP-specific humoral immune responses, including neutralizing antibodies, and cell-mediated immune responses in rhesus macaques. Monkeys immunized with HPV16L1 VLPs mounted a potent humoral response with strongly neutralizing antibodies and a strong L1-specific Th2 response as measured by IL-4 production by CD4+ T cells. Monkeys immunized with plasmid DNA or an adenoviral vector expressing HPV16L1 showed strong Th1/Tc1 responses as measured by IFN-gamma production by CD4+ and/or CD8+ T cells and potent humoral responses, but only weakly neutralizing antibodies. These data demonstrate that the nature of the immune response against HPV16L1 is dramatically different when it is introduced via different delivery systems. Additionally, these findings support the notion that an HPV16L1 VLP-based vaccine will induce the strongly neutralizing antibodies necessary for effective prophylaxis.

Sustained peptide-specific gamma interferon T-cell response in rhesus macaques immunized with human immunodeficiency virus gag DNA vaccines.

We examined the influence of dose and method of antigen delivery on the dynamics and durability of T-cell responses to candidate human immunodeficiency virus (HIV) vaccines. Codon-optimized sequences from the HIV gag gene were inserted into alternative DNA vaccine vectors to express the coding sequence with or without the tissue plasminogen activator leader sequence. We delivered the vaccines by intramuscular injection as plasmid DNA without adjuvant or as plasmid DNA formulated with a novel block copolymer adjuvant (CRL8623) and then monitored the ensuing T-cell responses by using a gamma interferon enzyme-linked immunospot assay. We demonstrated persistence of the cell-mediated immune (CMI) response in rhesus macaques for at least 18 months following a four-dose vaccination regimen. The plasmid vaccine, with or without CRL8623, was immunogenic in macaques; however, the form coadministered with adjuvant exhibited improved T-cell responses, with a bias toward more antigen-specific CD8(+) T cells. Finally, we examined the fine specificity of the T-cell response to the gag vaccines by testing the response of 23 vaccinated macaques to individual Gag 20-mer peptides. Collectively, the monkeys responded to 25 epitopes, and, on average, each monkey recognized a minimum of 2.7 epitopes. The results indicate that a broad and durable CMI response to HIV DNA vaccines can be induced in a relevant nonhuman primate model.