HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Kelly E Seaton
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Lamar Ballweber
Department of Obstetrics and Gynecology
University of Washington
Seattle, WashingtonUnited States of America

Audrey Lan
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Michele Donathan
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Sean Hughes
Department of Obstetrics and Gynecology
University of Washington
Seattle, WashingtonUnited States of America

Lucia Vojtech
Department of Obstetrics and Gynecology
University of Washington
Seattle, WashingtonUnited States of America

M Anthony Moody
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Hua-Xin Liao
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Barton F Haynes
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

Christine G Galloway
Vaccine and Infectious Disease Division
Fred Hutchinson Cancer Research Center
Seattle, Washington

United States of America

Barbra A Richardson
Vaccine and Infectious Disease Division
Fred Hutchinson Cancer Research Center
Seattle, Washington

United States of America

Department of Biostatistics
University of Washington
Seattle, WashingtonUnited States of America

Salim Abdool Karim
CAPRISA -Centre for the AIDS Programme of Research in South Africa
Nelson

Department of Epidemiology
Columbia University
New YorkNew York

United States of America

Charlene S Dezzutti
Department of Obstetrics, Gynecology and Reproductive Sciences
University of Pittsburgh School of Medicine
PittsburghPennsylvania

United States of America

M Juliana Mcelrath
Vaccine and Infectious Disease Division
Fred Hutchinson Cancer Research Center
Seattle, Washington

United States of America

Department of Medicine
University of Washington
Seattle, Washington

United States of America

Department of Laboratory Medicine
University of Washington
Seattle, Washington

United States of America

Department of Global Health
University of Washington
Seattle, WashingtonUnited States of America

Georgia D Tomaras
Duke Human Vaccine Institute
DurhamNorth Carolina

United States of America

*
Florian Hladik
Department of Obstetrics and Gynecology
University of Washington
Seattle, WashingtonUnited States of America

Vaccine and Infectious Disease Division
Fred Hutchinson Cancer Research Center
Seattle, Washington

United States of America

Department of Medicine
University of Washington
Seattle, Washington

United States of America

Mandela School of Medicine
University of KwaZulu-Natal
DurbanSouth Africa

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background: Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.Methods and Findings:We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.Conclusion: Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.

Introduction

Only approximately one in twenty to one in two thousand unprotected sexual encounters with an HIV-1-infected partner leads to systemic HIV-1 infection in the recipient [1,2]. There is evidence, though, that exposures to HIV-1 that fail to establish infection can still exert immunological effects. For example, HIV-1-specific T cells have been detected in highly HIV-1-exposed individuals who remain seronegative [3][4][5][6][7][8][9][10][11][12][13]. Some studies have also found mucosal HIV-1-specific neutralizing IgA in the cervicovaginal lavage and seminal fluid of HIV-1-negative individuals with HIV-1-infected partners [11,[14][15][16][17]. Such immunological responses may serve as markers of prior HIV-1 exposure. Risk status (i.e. level of HIV-1 exposure) is an important consideration when recruiting subjects for efficacy trials of HIV-1 prevention strategies -the lower the population risk of HIV-1 infection, the larger the study cohorts must be to conclusively test effectiveness. The presence of HIV-1-specific immunity at a site of potential HIV-1 transmission might also confer some protection from infection [18]. Understanding such immune responses could be of value for HIV-1 vaccine design.

HIV-1-specific antibody titers are in general easier to measure than HIV-1-specific T cell function, but there is little clear guidance regarding the optimal specimen types and sampling devices for the assessment of mucosal antibodies in HIV-1 vaccine and microbicide clinical trials. Cervicovaginal lavage (CVL) has been used for the quantification of proteins in genital secretions. However, CVL sampling is more complicated than swabbing, and the dilution of mucosal secretions and the resulting variability in recovered sample volumes can decrease the sensitivity of protein detection and pose a challenge for data interpretation [19][20][21].

Therefore in this study, we compared three direct vaginal sampling devices not requiring a lavage: Dacron swabs, flocked nylon swabs and Merocel sponges. Using a cohort of five HIV-positive and five HIV-negative women in Seattle, we determined total IgG and IgA concentrations obtained from each device, and then compared antibody reactivity to a panel of eight HIV-1 Env antigens with an HIV-1-specific binding antibody multiplex assay, previously utilized for both serum/plasma and mucosal specimens [22][23][24][25]. We then examined HIV-1-specific antibody responses in swabs obtained from African women participating in the control arm of the HPTN 035 microbicide trial, a Phase IIb study designed to assess the effectiveness of 0.5% PRO2000 gel and BufferGel for the prevention of HIV-1 infection [26]. We found HIV-1-specific IgA, but not IgG, antibodies in the vaginal secretions of nine of 57 HPTN 035 participants. These mucosal HIV-1-specific IgA antibodies could contribute to protection in these women or be a marker of another protective function; thus, defining mucosal HIV-specific antibodies in future microbicide trials should be of major interest to the HIV prevention field.

Methods

Clinical Cohorts and Ethical Approval

Two cohorts of women were enrolled in this study following review and all protocols were approved by the Fred Hutchinson Cancer Research Center Institutional Review Board, Duke University Medical Center IRB, and local IRBs at each of the HPTN 035 sites. All participants gave written informed consent to be a part of the study. The Seattle cohort included five HIVuninfected women at low risk for HIV-1 infection and five women with HIV-1 clade B infection who were on antiretroviral treatment (average blood CD4 count 837/ml). The African cohort comprised a total of 58 participants in the control, condom-use-only, arm of the microbicide trial HPTN 035 (ClinicalTrials.gov registration ID NCT00074425). Vaginal Dacron swabs from approximately 10 women from six HPTN 035 sites were included for analysis: Blantyre and Lilongwe, Malawi; Durban and Hlabisa, South Africa; and Harare and Chitungwiza, Zimbabwe. One of the 58 women was found to be clade C HIV-1 seropositive during the trial and is included here as a positive control. The demographics of the HPTN 035 study participants, the study protocol and the results of the clinical study are described elsewhere [26,27]. Vaginal smears were Gram stained for bacterial vaginosis (BV) diagnosis by Nugent scoring. Gram stains with a Nugent score of 4-6 or $7 were classified as signifying abnormal flora or BV, respectively.

Swab collection and processing in the Seattle cohort

In the Seattle cohort, three types of swabs were used for the collection of vaginal secretions: Dacron polyester-tipped swabs (Fisher Scientific Cat. No. 23-400-122), flocked nylon swabs (Copan Diagnostics; Fisher Scientific Cat. No. 23-600-957) and Merocel sponges (Merocel Eye Spears, Beaver-Visitec International, Item No. 400101). Six swabs were taken from each of five HIV-1-infected and five HIV-uninfected women during two sessions that were spaced one hour apart. Three swabs, one of each type, were collected in each session. Half of the women were randomly assigned to begin session 1 with Dacron swabs followed by flocked swabs; the other half began session 1 with flocked swabs followed by Dacron swabs. This swab order was reversed in session 2. In both sessions, Merocel sponges were the third and last sample type obtained. Swabs were placed into the posterior vaginal fornix until visibly saturated, but never longer than 1 minute. Swabs were immediately placed into 500 ml of elution buffer (cold phosphate-buffered saline (PBS) containing 1% protease inhibitor cocktail (Calbiochem Set I), 10% Igepal (Sigma) and 0.25% bovine serum albumin (Sigma)). After transport on ice to the laboratory, secretions were eluted on the same day. Swabs or Merocel sponges were transferred into a pre-chilled 0.45 mm SPIN-X filter (Corning Life Sciences). Swab or sponge handles were clipped off, the transport buffer was transferred to the SPIN-X filters, and the SPIN-X filters were spun at 13,0006g for 15 min at 4uC. 300 ml of elution buffer were added, the SPIN-X filters were incubated on ice for 5 min, and spun at 13,0006g for 30 min at 4uC. Filter and sponges were removed and discarded. Eluates were distributed at 100 ml per vial and stored at 280uC until assayed.

Swab collection and processing in HPTN 035

In HPTN 035, vaginal Dacron swabs were collected as described previously [27]. In brief, during a quarterly pelvic exam, a Dacron swab was applied to the posterior fornix of the vagina to saturate the tip with fluid and then placed in a cryovial containing 400 ml of PBS. The cryovials were stored at 280uC at the sites and shipped to the Microbicide Trials Network (MTN) Network Laboratory after the primary study results were available [26]. Swabs were thawed on wet ice, vortexed, and compressed against the side of the tube to elute secretions. Swabs were removed and placed upside down in a new tube and centrifuged at 7006g for 10 min at 4uC to elute remaining secretions. Eluates of both tubes were combined, using 200 ml of PBS for rinsing. The combined eluate was centrifuged again to pellet particles. Eluates (approximately 350 ml total) were stored in aliquots at 280uC until assayed. The HIV-1 binding antibody multiplex assay has been previously described [23][24][25][28][29][30]. Briefly, HIV-1 antigens were conjugated to polystyrene beads (Bio-Rad) and binding of IgG or IgA in the eluates to the bead-conjugated HIV-1 antigens was detected by mouse anti-human IgG-PE (Southern Biotech) or goat anti-human IgA-PE (Jackson Immunoresearch). To measure HIV-1-specific IgA, eluates were first depleted of IgG using Protein G high-performance MultiTrap plates (General Electric) according to the manufacturer's instructions. After incubation of beads and eluates, the beads were washed and acquired on a Bio-Plex 200 instrument (Bio-Rad). HIV-1-specific binding antibodies for each antigen were measured as Mean Fluorescence Intensity (MFI). Total IgG and IgA antibody measurements for calculating specific activity were performed using a Bio-Plex Pro Human Isotyping 7plex panel (Bio-Rad) according to the manufacturer's instructions.

Positivity calls per HIV-1 antigen per antibody isotype were based on preset criteria determined from HIV-1 negative samples in the Seattle cohort and were consistent with historical positivity criteria for mucosal responses. An HIV-1-specific antibody response was considered positive if it met the positivity criteria for both MFI and specific activity. Positive MFI responses were defined as greater than the mean MFI plus 5 standard deviations of the antigen with the highest background among the 30 samples taken from the five uninfected women in Seattle. To account for subject-to-subject variation in fluid collected (including levels affected by timing of the menstrual cycle and contraceptive use), MFI values were normalized to total IgG or IgA and computed as specific activity (antigen specific MFI/ng ml 21 total IgG or IgA). All total antibody measurements were assesed in samples diluted 1:3 in assay diluent (Bio-Rad). Positive specific activity responses were defined as greater than the mean plus five standard deviations of the specific activities measured in the five seronegative individuals in the Seattle cohort.

Reagents for antibody measurements

Antibody responses were determined for the following antigens: Clade B gp41 Immunodominant epitope tetramer (gp41 ID epitope) (Biotin -CRVLAVERYLRDQQLLGIWGCSGKLICT-TAVPWNASWSNKSLNKI), 2F5 mAb epitope tetramer (Biotin-GGGQQEKNEQELLELDKWASLWN), clade B MPER tetramer (MPR.03) (KKKNEQELLELDKWASLWNWFDITNWL-WYIRKKK-Biotin) (containing the linear binding sites for MPER neutralizing antibodies), and clade C MPER (MPR.C956) tetramer (Biotin-NEQELLELDKWASLWNWFNITNWLW) (derived from a clade C sequence and containing the linear binding sites MPER neutralizing antibodies (tetramers provided by Dr. Anthony Moody, Duke Human Vaccine Institute; [31,32]); Consensus gp120 Env (Con6 gp120), clade C gp140 Env trimer (1086 Trimer) (a transmitted/founder sequence envelope sequence from a clade C infected patient from Malawi, [33] and a Group M consensus gp140 Env (ConS gp140) [34][35][36] (envelope proteins provided by Drs. Barton Haynes and Larry Liao, Duke Human Vaccine Institute). The consensus sequence for Group M is central to all circulating clades, and reacts well with sera from all subtypes [34][35][36] and was found to be similar to autologous envelopes in detection of the initial antibody response in acute infections [23].

Statistical analysis

For the Seattle pilot cohort, ANOVA with fixed effects was applied to assess the influence of swab type, time point of sample collection and subject HIV serostatus on log 10 total IgG and IgA concentrations. ANOVA with fixed effects was also used to assess the influence of swab type and time point of sample collection on log 10 HIV-1-specific IgG activities. For the HPTN 035 cohort, the effect of study country or vaginal flora Nugent scoring on the presence of vaginal HIV-1-reactive IgA antibodies was tested by Chi-square test or Fisher's exact test, respectively.

Results

Comparison of three sampling devices for measuring total and HIV-1 envelope-specific IgG and IgA antibodies in vaginal secretions

Dacron polyester-tipped swabs, flocked nylon swabs and Merocel sponges were obtained from 5 HIV-1 infected women and 5 HIV uninfected women (Seattle cohort). Six samples were taken from each woman, one of each type in a first session, and one of each type again one hour later. The amount of total IgG and IgA collected from each sampling device did not differ by device type (p = 0.7 for IgG; p = 0.8 for IgA) or time point of sampling (ANOVA with fixed effects, p = 0.6 for IgG; p = 0.4 for IgA) ( Figure 1A). The total amount of IgG also did not differ by HIV serostatus (p = 0.5), however the total amount of IgA was significantly higher in the HIV-negative subjects (mean log 10 4.24 ng/ml60.65 versus log 10 3.9160.48; p = 0.03) ( Figure 1A). In these samples, we additionally examined HIV-1-specific activity against a panel of eight Env antigens covering multiple epitopes in gp41, the gp41 membrane proximal external region (MPER), gp120 and gp140. HIV-1-specific IgG activity for each antigen did not differ by device type or time point of sampling (all F-test p values.0.05). There were too few positive values to statistically evaluate differences in HIV-1-specific IgA activity. Specific activities for gp41-binding IgG ( Figure 1B, left panel) and IgA ( Figure 1B, right panel) are shown as a representative antigen. In conclusion, Dacron swabs, flocked swabs and Merocel sponges appear equally suitable for measuring total immunoglobulins and HIV-1-specific IgG and IgA in vaginal secretions.

Comparison of HIV-1 envelope-specific IgG to IgA responses in the vagina of HIV-1 infected women

HIV-1 infection induced a broad range of envelope-specific IgG responses in the vaginal mucosa of the five HIV-1 infected women in Seattle (Figure 2A and B). These responses favored gp41 and the gp41 ID epitope (5/5 women tested positive); however significant responses were also seen against the gp120 antigen Con6 gp120 (3/5), and the gp140 antigens Clade C 1086 trimer (4/5) and ConS gp140 (4/5). Only one woman had IgG antibodies positive responses, with the colors corresponding to specific individuals. Samples that did not meet the positivity criteria are displayed at zero for specific activity. against the three membrane proximal external region (MPER) antigens. IgA responses were seen at a lower frequency ( Figure 2C and D). HIV-1-specific IgA activity was predominantly directed toward gp41 (4/5 women) and the gp41 ID epitope (2/5). One woman had gp140-specific IgA against the Clade C 1086 trimer and ConS gp140 antigens. None of the women had vaginal IgA antibodies specific to the three MPER antigens or to gp120. None of the 30 specimens taken from the five HIV uninfected women had IgG or IgA antibodies to any of the tested HIV-1 antigens (not shown). These data indicate that the HIV-1 binding antibody multiplex assay detects vaginal HIV-1 specific antibodies in a highly specific manner, and that the vaginal IgA response to HIV-1 infection is markedly narrower than the vaginal IgG response.

HIV-1 envelope-specific binding antibody responses in the vaginal mucosa of African women participating in the HPTN 035 microbicide trial

Having demonstrated the specificity of our assay to detect HIV-1-reactive antibodies in vaginal secretions, as well as the suitability of Dacron swabs to obtain secretions for performing these measurements, we next examined vaginal HIV-1-specific antibody responses in a cohort of African women enrolled in the HPTN 035 Phase IIb clinical trial. Of the 58 women we analyzed from this cohort, 57 women remained uninfected while one woman seroconverted. Vaginal secretions from the sole seroconverter contained HIV-1-specific IgG ( Figure 3A) but not IgA antibodies ( Figure 3B). Pooled HIV + immunoglobulin, as well as the control IgG antibodies 4E10 and 2F5, showed binding to their respective antigens ( Figure 3A). Likewise, the control IgA antibodies 7B2, b12 and 2F5 bound their respective antigens ( Figure 3B).

None of the 57 HIV uninfected women had HIV-1 envelopereactive vaginal IgG antibodies ( Figure 3C). Nine women (15.8%) had HIV-1 envelope-reactive IgA antibodies in their vaginal secretions ( Figure 3D and E). IgA antibodies to both Con6 gp120 and ConS gp140 were detected in six women (10.5%), and IgA antibodies from two additional women were specific for the gp41 ID epitope (3.5%). Secretions from one of these two women also reacted to the MPR.03 epitope (1.8%). Vaginal secretions from the ninth woman reacted to the 2F5 epitope (1.8%). These data indicate the presence of HIV-1 envelope-reactive IgA but not IgG antibodies in vaginal secretions of ,16% of African women participating in the control arm of the HPTN 035 microbicide trial. While antibodies from each woman reacted to only one or two HIV-1 antigens, collectively, antibodies from the nine IgA-positive women reacted to five of the eight HIV-1 envelope antigens tested.

Relationship of vaginal HIV-1-reactive IgA antibodies in HIV-negative women to HPTN 035 study site and country and to bacterial vaginosis

Particularly high HIV-1 incidence rates have been reported for South Africa, with a reported prevalence of 21.1% in young women aged 20-24 (2008), and a prevalence up to 39.5% in pregnant women in KwaZulu Natal (2010) [37,38]. Consequently, the likely higher HIV-1 exposure rates in South Africa could lead to the emergence of vaginal HIV-1-reactive IgA antibodies more often than in countries with lower HIV-1 incidence. We thus investigated the geographic distribution of the nine women with vaginal HIV-1-reactive IgA antibodies were seen. In South Africa, 3/9 women in Durban (33.3%) and 1/9 women in Hlabisa (11.1%); in Zimbabwe, 3/10 women in Chitungwiza (30%) and 0/ 10 women in Harare (0%); and in Malawi, 1/9 women in Lilongwe (11.1%) and 1/10 women in Blantyre (10%), were IgA positive. Thus, 22.2% of South African, 15% of Zimbabwean and 10.5% of Malawian women tested positive for vaginal HIV-1reactive IgA antibodies, with no significant differences between countries (Chi square p = 0.62).

We also investigated whether a relationship between vaginal HIV-1-reactive IgA status and bacterial vaginosis (BV) existed. Twenty of the 57 HIV-1 uninfected HPTN 035 women had abnormal vaginal flora (Nugent score 4-6) and seven had BV (Nugent score$7). Among the nine women with HIV-1-reactive IgA antibodies, two had abnormal flora and one had BV, while 18 of the other 48 women had abnormal flora (Fisher's exact p = 0.47) and six had BV (p = 1.0). Thus, abnormal vaginal flora and bacterial vaginosis did not correlate with the presence of vaginal HIV-1 envelope-reactive IgA antibodies.

Discussion

Our study confirms a number of prior reports of HIV-1-specific IgA in the genital tracts of HIV-1-exposed seronegative women [6,14,17,[39][40][41][42]. Between 38% and 82% of highly exposed seronegative (HESN) women [6,41,42] have been reported to have genital HIV-1-specific IgA, although one study of uninfected sex workers from the Gambia failed to detect any vaginal antibody responses against HIV-1 [43]. In our study, 9/57 (15.8%) women had vaginal HIV-1-specific IgA antibodies. Reasons for divergent frequencies in different studies and cohorts have been discussed elsewhere [16], but actual exposure rates to HIV-1 should be a major determinant. It is not surprising that IgA frequencies in our cohort were at the lower end of the reported range, because HPTN 035 study subjects [26], including the 57 women we studied, were not screened for self-reported risk factors such as frequent unprotected sex, a known HIV-infected partner and/or commercial sex work. Thus, HPTN 035 study women did not fit the HESN definition [44,45] and their risk profile was on average likely lower than that of HESN individuals. Moreover, sample collection and storage methods, in addition to the conservative, two-tiered definition of antibody positivity, may have contributed to the relatively low frequency of HPTN 035 women with positive HIV-1-specific vaginal IgA. Additionally the hormonal cycle can influence both IgG and IgA antibody levels [46,47]. One limitation of our study with HPTN 035 samples was that due to study size and inclusion criteria (,3099 female participants with some contraceptive use) the sample collections were not synchro-nized around the participant's menstrual cycle. We controlled for antibody levels among participants in our analyses by factoring in total antibody levels along with the HIV-1 specific antibody calculations.

Nevertheless, vaginal swabs from ,16% of the uninfected HPTN 035 women we tested were positive for HIV-1-specific IgA antibodies, whereas none of the 57 women were positive for HIV-1-specific IgG antibodies. This indicates that the presence of HIV-1-specific mucosal IgA could be used as an indicator of HIV-1 exposure in subjects participating in HIV prevention trials [48]. Additionally, our study demonstrated that vaginal swabs routinely obtained during a microbicide trial and stored frozen for several years can be used for that purpose. While this was the first time that HIV-1-specific mucosal antibodies were evaluated in a microbicide trial, similar studies should be pursued in ongoing and future trials of topical pre-exposure prophylaxis (PrEP). Of particular interest will be the question of whether efficacious topical PrEP, which as of yet has not been developed, might contribute to a mucosal state of HIV-1-specific immunity by exposures to microbicide-inactivated HIV-1 and/or by aborting local infections. This principle has been demonstrated in macaques [49] and may over time provide an added benefit to using topical PrEP, i.e., an additional level of resistance through adaptive immunity. The nature of this immune response in microbicide-using women would be of major interest to the HIV vaccine field.

In this context, it is interesting to note that antibody responses in HIV-1 infected persons are dominated by IgG, whereas HIV-1specific IgA appears to be impaired [25,50,51]. In contrast, exposed uninfected individuals develop nearly exclusively HIV-1specific antibodies of the IgA type [16], as also seen in our study here. Furthermore, we have previously demonstrated that the initial antibody response to HIV-1 infection is directed to gp41 [23] and that the initial mucosal IgA response (in cervical lavage and seminal plasma) is also directed to gp41, with limited reactivity to other regions of the HIV-1 envelope [25]. In the exposed uninfected HPTN 035 women, however, six of the nine women with positive HIV-1-specific IgA in the vagina showed reactivity to gp120 and gp140, whereas only three reacted to gp41 epitopes. This finding is consistent with our work in another exposed uninfected cohort (CHAVI 002; United Kingdom and Uganda), where we detected anti-HIV-1 gp120 IgA, but not IgG, responses in blood (Shen et al., unpublished). Thus, the results from both studies, with samples obtained from different geographic locations, converge on the same notion that HIV-1 gp120-specific IgA are induced in individuals that are (likely repeatedly) exposed to HIV-1, but are not part of the initial response to acute HIV-1 infection. Due to low numbers of women with mucosal HIV-1 specific IgA responses, we did not have the power to robustly determine if there was an association between sexual risk factors and the presence of vaginal HIV-1 IgA. The reasons for this skewing of genital HIV-1specific antibody responses in exposed uninfected women toward the IgA subtype and toward HIV-1 gp120/gp140 remain unclear. However, these findings emphasize that further study of mucosal HIV-1-specific IgA responses in exposed uninfected individuals could uncover clues for purposefully driving a vaccine-induced response toward protective IgA-mediated mucosal immunity.

One caveat of the current study is that limited sample volume precluded testing of these mucosal antibodies for specific antiviral functions. Thus, we were not able to determine if the detected antibodies can block HIV-1 infection. Further studies comparing other methods for antibody elutions from wecks [52,53] may provide improved antibody recovery. Other investigators have shown that monoclonal HIV-1 gp41-specific IgA antibodies derived from highly exposed seronegative individuals can neutralize HIV-1 and inhibit virion transcytosis through epithelial cells [40,54,55]. It is also encouraging that protection from simian/ human immunodeficiency virus infection in macaques can in principle be achieved by passive administration of neutralizing and non-neutralizing HIV-1 inhibitory antibodies to the vagina [56,57]. Thus, further studies are needed that collect larger volumes of mucosal fluids to continue investigations into the antiviral properties of mucosal IgA in HIV-1 exposed uninfected individuals, such as antibody-mediated movement arrest of HIV-1 in cervical mucus [58], virion capture [30,59] and neutralization [11,14,16,17].

Evaluation of mucosal immune responses is also crucial to interpret the findings of HIV-1 vaccine trials. The lack of such mucosal studies has left us with unanswered questions regarding the lack of efficacy of the HIV-1 vaccines tested in the Step, Phambili and HVTN 505 trials [60][61][62][63], as well as other potential correlates of protection conferred by the HIV-1 vaccine tested in the RV144 trial [64,65]. These trials did not assess mucosal immune responses largely because of the lack of optimized and standardized procedures for mucosal specimen harvesting, including uncertainty over which specimens and sampling devices are best for a given research question. This is in part due to the variability of methods used to collect genital secretions, yielding different amounts of fluid and leading to sample dilution when using cervicovaginal lavage [19][20][21]. We compared three devices for harvesting undiluted vaginal secretions and found that Merocel sponges were preferred by clinicians and recovered similar amounts of total and HIV-1-specific antibodies as compared to Dacron and flocked nylon swabs. Thus, all three sampling devices could be used in future vaccine and topical PrEP trials where measurement of vaginal HIV-1-specific antibodies is desired.

Taken together, we report that genital HIV-1 envelope-specific antibodies in HIV exposed uninfected women in a large microbicide trial were predominantly of the IgA isotype. Strikingly, the epitope specificities of these Env-specific IgA were predominantly directed to gp120, unlike those IgAs induced in acute HIV-1 infection that predominantly target gp41 [23,25]. Further study of these responses to determine their usefulness as markers of HIV-1 exposure as well as their protective potential is warranted.

Figure 1 .
1Total and HIV-1-specific IgG and IgA in vaginal secretions do not differ by sampling device or timepoint in the same women. (A) Dacron swabs, flocked swabs, and Merocel sponges were collected from 5 HIV-1 infected (colored circles) and 5 HIV uninfected (grey triangles) women and the eluate was analyzed for total IgG (left) and total IgA (right). Symbols represent the average of two collection time points spaced one hour apart, for each woman and sampling device. For HIV-1 infected women, colors indicate specific individuals. (B) Samples from the first time point (filled circles) and one hour later (open circles) from the 5 HIV-1 infected women were analyzed for gp41-specific IgG (left) and IgA (right) activity, calculated as antigen-specific mean fluorescence intensity (MFI)/ng ml 21 total IgG or IgA. Colors indicate specific individuals. The box-andwhisker plots represent median, 25 th and 75 th percentiles, and range. doi:10.1371/journal.pone.0101863.g001

Figure 2 .
2HIV-1 infection induces a broader range of HIV-1 Envspecific IgG than IgA antibodies in vaginal secretions. Vaginal secretions were collected from 5 HIV-1 infected women and analyzed for IgG (A-B) and IgA (C-D) binding to the indicated HIV-1 Env antigens. Antigens not specified as Clade C or consensus (Con6 and ConS) are derived from Clade B viruses. (A) Average HIV-1 antigen-specific IgG activity from the combination of the three sampling devices and two time points, calculated as antigen-specific MFI/ng ml 21 total IgG. Black symbols represent negative responses and colored symbols indicate Multiplex assay for detection of HIV-1-binding antibodies

(B) Percentage of women with positive IgG binding. (C) Average HIV-1 antigen-specific IgA activity, calculated as antigen-specific MFI/ng ml 21 total IgA. (D) Percentage of women with positive IgA binding. doi:10.1371/journal.pone.0101863.g002

Figure 3 .
3HIV-1 exposure elicits anti-Env IgA but not IgG antibodies in the vaginal mucosa. Vaginal Dacron swabs were collected from 57 HIV seronegative women enrolled in the HPTN 035 microbicide trial and analyzed for IgG (C) and IgA (D-E) binding to the indicated HIV-1 Env antigens. (A) Positive IgG controls. Binding of purified pooled HIV-1 + immunoglobulin (HIVIG), 4E10 monoclonal IgG (50 mg/ml), 2F5 monoclonal IgG (16 mg/ml) and swabs from an HIV-1 infected HPTN 035 participant (HIV + ) in mean fluorescent intensity (MFI). (B) Positive IgA controls. Binding of 7B2 monoclonal IgA (1 mg/ml), b12 monoclonal IgA (20 mg/ml) and 2F5 monoclonal IgA (1 mg/ml) in MFI. Lack of IgA binding from an HIV-1 infected HPTN 035 participant (HIV + ) is also shown. (C) HIV-1 antigen-specific IgG activity, calculated as antigen-specific MFI/ng ml 21 total IgG, in 57 women participating in HPTN 035. One additional HIV-1 infected HPTN 035 participant (HIV + ) is also shown. (D) HIV-1 antigen-specific IgA activity, calculated as antigen-specific MFI/ng ml 1 total IgA. For the 57 HPTN 035 women (squares), black symbols signify negative responses and colored symbols signify positive responses, with colors indicating specific women. Samples that did not meet the positivity criteria are displayed at zero for specific activity. One additional HIV-1 infected HPTN 035 participant (HIV + ) is also shown (open circles). (E) Percentage of the 57 HPTN 035 women with positive IgA binding. doi:10.1371/journal.pone.0101863.g003
July 2014 | Volume 9 | Issue 7 | e101863
PLOS ONE | www.plosone.org
AcknowledgmentsThe authors would like to thank Stephen Voght for critical reading and editing of the manuscript, and the study participants in Seattle for volunteering for this study. We thank R. Glenn Overman, Vicki Ashley and Judith T. Lucas for technical assistance with the antibody assays.
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Africa's largest long-lasting insecticide-treated net producer: lessons from A to Z Textiles

Hassan Masum
Ronak Shah
Karl Schroeder
Abdallah S Daar
Peter A Singer
Africa's largest long-lasting insecticide-treated net producer: lessons from A to Z Textiles
R E S E A R C H Open Access
Background: Field trials have demonstrated the efficacy of insecticide-treated nets, and the WHO has recently endorsed a shift toward Long-Lasting Insecticide Treated nets (LLINs) due to factors such as reduced distribution costs. However, the need for LLINs poses several challenges. Is it possible to manufacture LLINs in large quantities in the African continent, where malaria is most endemic? When production is located in low-income countries, what role is played by local funding and employment, scaling up manufacturing, and partnerships? What factors influence availability and pricing? Discussion: A case study of A to Z Textiles was undertaken to answer the question of how large-scale production of LLINs can occur in a low income setting. One of the largest sources of bed nets for Africa, A to Z Textiles is Africa-based, and its Tanzanian operations have a production capacity of 30 million LLINs per year, along with full WHO recommendation for its nets. Our analysis is based on semi-structured interviews with key informants familiar with A to Z, site visits in Tanzania, and literature reviews. This paper discusses the history and current status of A to Z Textiles, identifies the factors that led to its success, and suggests policy considerations that could support similar initiatives in the future. Local funding, scaling up manufacturing, technology transfer, and partnerships all played important roles in A to Z's ascent, as did perceived benefits of local employment and capacity-building. Regulatory issues and procurement rules acted as barriers. A to Z cost-effectively manufactures high-quality LLINs where malaria is most endemic.Summary: With a production capacity of 30 million LLINs per year, and full WHOPES (WHO Pesticide Evaluation Scheme) certification, A to Z Textiles demonstrates how key health goods can be successfully produced in the lowincome countries that use them. Its example may be instructive and of high interest to readers in the malaria community, especially in developing countries, and to those who wish to support or partner with efforts by developing countries to build their health innovation capacity.

Background

Most of the malaria disease burden is in Africa, with a cost in human life of around one million deaths per year, of which a large majority are children [1]. Among several approaches such as vector control strategies that are being pursued to prevent malaria, insecticide-treated nets (ITNs) are one of the best current public health interventions in terms of safety and cost-effectiveness [1]. Large-scale studies have documented the beneficial impact of ITNs on reducing malaria mortality and morbidity, including infant mortality [2,3].

More recently, a shift toward Long-Lasting Insecticide-treated Nets (LLINs) has been endorsed by the WHO Global Malaria Programme (WHO/GMP) [4]. LLINs are made with material that has insecticide bound around or incorporated within the fibers. They must retain effectiveness for at least 20 WHO standard washes (i.e. laboratory procedures that simulate usage) and three years of use in field conditions. LLINs reduce the need for retreatment and replacement of nets, offer longer protection for users, and help reduce distribution and maintenance costs.

While the reasons for preferring LLINs are clear, the production of LLINs poses several challenges. Is it possible to manufacture them in large quantities in the African continent, where malaria is most endemic? When production is located in low-income countries, what role is played by local funding and employment, scaling up manufacturing, and partnerships? How do regulatory issues and procurement rules affect the viability of LLIN production? What factors influence availability and pricing?

These issues are illustrated in the case of Africa's largest bed net manufacturer, A to Z Textilesa familybased Tanzanian enterprise that grew from humble beginnings to become a major bed net manufacturer. A to Z illustrates the role of African entrepreneurship in successful technology transfer and development. It shows how supportive partnerships can mobilize the South's capacity to tackle its own health challenges.

We used a case study design. Our analysis is based on semi-structured interviews with key informants familiar with A to Z, site visits in Tanzania, and literature analysis of background documents on A to Z, related articles from the peer-reviewed literature, news reports, books, web sites, and relevant reports from WHO (the World Health Organization) and other groups. We conducted interviews with personnel of A to Z, and with PSI (Population Services International), Acumen Fund, and Tanzanian government and health organizations, all of whom interacted with A to Z in its bed net endeavors. A to Z was asked to fact-check the case study; the analysis and interpretation is our own. All quotes are from the interviews unless otherwise noted, and with permission. This study was approved by the Office of Research Ethics of the University of Toronto.

In this article, we summarize the history and current status of A to Z Textiles, and analyze the factors that led to its success. We then suggest policy considerations that could further support similar producers of health goods in low income settings in the future.

Discussion

A chance meeting

A to Z Textiles is based in Arusha, Tanzania. The company started in 1965 with five sewing machines run by family members of the late N.H. Shah (father of the current CEO Anuj Shah). The family initially made garments for the local market, and in 1978 they started producing bed nets.

In 1998, Professor Don De Savigny, then with Canada's International Development Research Centre, was visiting Arusha with Dr. Hassan Mshinda, then director of Tanzania's Ifakara Health Research Institute. They had come to meet the owners of Sun Flag, a local company that made bed nets.

While insecticide-treated bed nets were not new, they had a significant problem: they needed to be regularly re-dosed with insecticide. The companies that made the insecticide preferred to sell it by the barrel, and the consumers who bought the nets often had more urgent priorities than bringing the nets to a central depot to be re-treated. De Savigny hoped Sun Flag would partner with an initiative from PSI (Population Services International, a non governmental organization with global operations) to provide home treatment kits that people could use to dose the nets themselves.

Sun Flag wasn't interested, and De Savigny and Mshinda found themselves back on the street. Dr. Mshinda pointed out that there was another bed net manufacturer across the road: a 33-year old family-run company called A to Z Textiles. With nothing to lose, the two men knocked on the gate at A to Z. They were met by Anuj Shah and his Production Director, Binesh Haria.

Opportunity and pitch

The timing of De Savigny and Mshinda's visit might not have been right for Sun Flag, but it was perfect for A to Z. De Savigny and Mshinda were looking for ways to alleviate the suffering caused by malaria, and when they pitched the home treatment kit as a business opportunity, Anuj Shah immediately saw its potential.

By the mid 1990s, their business had been facing challenges for years because trade liberalization in the 1980s had resulted in a large influx of cheaper second hand clothing into the local Tanzanian garments market. A to Z already made bed nets, but neither Anuj nor Binesh had guessed how important nets could be in the fight against malaria.

According to De Savigny, he told Anuj Shah, "If you get into this, you could steal the margin on this, because the public sector is about to start promoting this product." He pointed out that public sector institutions were never going to promote A to Z's baseball caps or T-shirts, "but they will promote this one, and if you have a better net [than Sun Flag], you're going to get the business."

This first meeting with A to Z was cordial, but as De Savigny put it, "We thought, well, it's another one of these factories and they're not going to listen to us…it was a very polite meeting and we got their attention, but we didn't think too much about this afterward." De Savigny and Mshinda left Arusha to continue promoting the home kits for re-dosing ITNs elsewhere. Six months after that first encounter, a meeting was held in Dar es Salaam by Canada's International Development Research Centre, PSI, and Health Bridge Canada. The purpose of the meeting was to bring the public sector, private sector, and NGOs together to explore ways to engage both the public and private sectors in rolling out ITNs across Tanzania. A to Z was invited to the meeting, but no one expected them to come. Professor De Savigny describes what happened:

"We had a reception to kick off the meeting and we had the Chamber of Commerce, the various Ministries, and the high level officials from the Ministry of Health… we made an exhibition space in the reception area for the insecticide manufacturers [who were] by then making kits, and for any net manufacturers who wanted to show up. So Sun Flag came and they hung up their standard white conical net on this white wall. And just before the speeches started, in came Binesh and Anuj. As we said, we never thought we'd see these people again, and they walked in and they hung up their very first six foot by six foot green, silky rectangular net -[a] huge net, and they hung it up on the wall and everybody flocked over there to see this fantastic new, domestic net. AMREF, the African Medical Research Foundation, was there from Nairobi and they went up and they gave an order for 100,000 on the spot."

From Arusha to Tokyo

By 2003 A to Z was the largest producer of conventional (i.e. non long-lasting) insecticide impregnated nets in Africa, producing over 6 million nets annually. This success was enabled by A to Z's strengths in plastics, textiles, and production technologies, which positioned A to Z to scale up production and adopt new technologies when Sumitomo Chemical Company of Japan presented them with an even bigger opportunity.

Sumitomo was founded in 1913, and has grown to be a multinational organization with over 100 companies in sectors including basic chemicals, petrochemicals, fine chemicals, and pharmaceuticals. Since the 1980s, scientists at Sumitomo had been developing insect-repellent products. Their LLINs had insecticide incorporated into the fiber of the net, which allowed them to release insecticide for up to 5 years. Primarily a chemical company, Sumitomo lacked expertise in packaging, marketing, and distribution in the developing world, and was searching for a partner.

In 2002 the World Health Organization (WHO) and the Roll Back Malaria Partnership approached Sumitomo, encouraging the company to look into manufacturing "Olyset" LLINs in Africa. A to Z was one company short-listed by WHO for Sumitomo to visit, and was deemed an attractive candidate with expertise in both textiles and plastics.

The dynamic synergy between A to Z Textiles and Sumitomo led to a royalty-free technology transfer of Olyset LLIN technology to A to Z. The technology transfer was facilitated by WHO, the social venture capital group Acumen Fund, and the Roll Back Malaria Partnership [5]. Dr. Mshinda further explains the terms of the transfer:

"The technology transfer entails that the raw material with the insecticide inside is provided by Sumitomo in its pellet form, along with their expertise around the technology. A to Z's expertise lies in manufacturing this product. Sumitomo is basically a chemical company, so they do not have that expertise in manufacturing. The actual process of incorporating the insecticide remains with Sumitomo, which is a [trade] secret." A to Z also produces textiles, plastics, and bottled water. In 2009, LLINs accounted for roughly 80% of total revenue and 90-95% of production. The joint venture with Sumitomo dedicated to Olyset production was set up in 2005 as an affiliated company, Vector Health International, in which Sumitomo and A to Z reportedly made equal initial co-investments of $7.5 million.

As of 2010, A to Z was a profitable enterprise employing over 7000 staff. Their annual production of 25 million nets was projected to increase to 30 million nets during 2010. Only 5% of A to Z's output goes to private buyers, mainly the middle and upper class. A to Z's biggest non-private buyers are organizations like UNICEF, and the governments of Tanzania, Mozambique, Malawi and Kenya, which have a variety of distribution models and subsidization schemes to deliver nets to end users.

A to Z anticipates a total need of around 280 million bed nets in Africa over the next three years; i.e. a market demand of approximately 85 million nets per year. Of these, A to Z is projected to produce roughly 30 million Olyset nets annually in partnership with Sumitomo, and Sumitomo is projected to produce an additional 21 million Olyset nets in facilities based in China and Vietnam.

By way of comparison, according to the World Malaria Report 2009, in 2008 manufacturers cumulatively reported a total of 57 million bed nets delivered to African countries, and ministries of health cumulatively reported a total of 38 million bed nets distributed within African countries [6]. (The report suggests that the difference between these two figures could be due to the lag between delivery and distribution, inadequate record-keeping, distribution by the private sector that is not included in ministry of health data, or other unknown factors.)

The Olyset net was the first LLIN to gain full WHO recommendation under the WHO Pesticide Evaluation Scheme (WHOPES), in October 2001 [7]. It was one of only two LLINs to have achieved full WHO recommendation as of August 2009, the other being the Vestergaard Frandsen Permanet 2.0 [8]. (The latter was reported in company documents to be manufactured in Vietnam and Thailand, and have a cumulative total of over 170 million distributed worldwide.) Olyset is the only LLIN with full WHO recommendation being manufactured in Africa.

There is evidence that Olyset nets retain some efficacy even after seven years in the field [9,10]. However, after a review of available evidence in 2009, the WHOPES Working Group decided not to extend WHO full recommendation from 3 to 5 years [11], indicating that the evidence could not prove that sufficient efficacy was maintained in the 4 th and 5 th years.

We turn now to a discussion of what helped A to Z succeed, and what their example suggests for future development of health solutions in low-resource settings.

Lessons learned Family funding and entrepreneurship

A to Z's holdings are primarily family-ownedthe Shah family has invested millions of dollars, making them the biggest investor along with Sumitomo. Family financing is a common formula in African and South Asian business ventures, especially when alternative risk capital is absent, as it can provide both capital and "smart money" that knows what ventures are feasible in the local environment [12]. While family financing holds long-term risks from infighting, loss of competence, and corruption, evidence of this was not found in the case of A to Z, and family firms dominate in most countries with short industrial histories [13].

The fact that the Shahs chose to take the risk of investing in LLIN production was itself a key decision point; the chance meeting with De Savigny and Mshinda in 1998 was key to making this opportunity apparent. Similarly, the Sumitomo partnership for Olyset production was a second key decision point, in which several partners assisted [5]. These two decision points may illustrate the potential benefit to future funders and entrepreneurs of "global health market intelligence" that makes key opportunities more apparent.

Manufacturing at scale and partnerships

A to Z has been instrumental in bringing manufacturing to scale in Tanzania while addressing an important health problem. There appear to be three main reasons why A to Z has been able to go from manufacturing 300 000 LLINs per year in 2003 to 25 million per year in 2009, with a projected 30 million per year in 2010.

First, by the time it had begun discussions with Roll Back Malaria and Sumitomo around LLINs, it was already the largest African manufacturer of ITN nets, producing over 6 million nets by 2003. This prior manufacturing experience was critical in navigating the 2004 Olyset manufacturing ramp-up.

Secondly, A to Z had developed a strong partnership with Sumitomo that not only provided the technology, but also provided financial support through their joint venture. Without the Sumitomo partnership and LLIN technology, A to Z would have experienced difficulty in achieving WHO certification and consequent sales; however, without A to Z's strengths in distribution and local manufacturing, Sumitomo likely could not have achieved the African market success it has. Several parties were involved in making A to Z's partnerships happen, including WHO, PSI, and Acumen Fund [5]. A to Z itself understood and exploited potential partnerships, most notably in the partnership with Sumitomo for Olyset production. Such relationships can provide incentives and de-risking for the private sector in the developing world to engage in innovation and address health challenges, by making opportunities clearer and by bringing together organizations with complementary capacities. (Indeed, there may be value to doing this sort of matchmaking more systematicallyboth for Southern enterprises looking to get into the health field, and for Northern organizations wishing to find capable and reliable Southern partners [14].)

Finally, A to Z benefited from commitment and large demand from buyers, mainly the donor community. About 95% of production goes to non-private buyers, such as UNICEF, the Global Fund to Fight AIDS, Malaria and Tuberculosis, the President's Malaria Initiative, PSI, and African governments. In the absence of donor and public funding, i.e. relying solely on local private buyers, A to Z would have had to lower volume sold, pricing, or bothsuggesting the catalytic role that donor and public funding can play in scaling up Southern manufacturing. It remains to be seen whether local demand (both public and private) will supplant international funding over time. To support similar future ventures, it may be helpful to study the de-risking role to producers of commitments from public buyers made in advance of production, in order to entice the private sector into areas of public health they would normally not enter [15].

Distribution and marketing

A to Z has used three linked distribution channels. First, a traditional private distribution network, done through an extensive network of wholesalers and retailers in eastern and southern Africa. Second, a mobile distribution system composed of a fleet of 150 trucks within Tanzania, which is supplemented by trucks hired from transport companies if demand warrants, and reaches countries surrounding Tanzania including Kenya, Uganda, Zambia, Zimbabwe, Mozambique, Burundi, and Rwanda. (One 40-foot container holds 26 000 nets.)

Third, for their public sector buyers, A to Z delivers the nets to designated warehouses or the appropriate Ministries of Health, building on their private and mobile distribution systems. This represents a significant advantage over their international competitors, who may only deliver to national ports. Looking ahead, there may be opportunities for manufacturers like A to Z to expand their logistics support to public sector buyers and NGOs, in areas such as training users or monitoring bednet replacement statistics.

Regulation and tendering

Being one of only two LLINs to gain full WHO approval has made Olyset a preferred product for buyers. At the same time, Anuj Shah notes that some tenders for bednets require only interim WHO certification, for which there are more competitors (six as of August 2009) [8]. Even though bednets with the full certification are supported by a greater evidence base from field trials, this distinction has not been implemented by some buyers in the tendering process, which may put A to Z and other fully-certified manufacturers at a competitive disadvantage if they invest more in quality.

Some tenders for LLINs evaluate bids only on the cost to the port of shipping. Anuj Shah suggests requiring full certification in tenders, and using the price to village as the tender price: "I think the responsibility of delivering the nets to the village should be on the manufacturers. Donor should say that you deliver. How you deliver is up to you." Using "full-cost" pricing could be a win-win situation, with buyers and funders able to evaluate long-term pricing for the provision of malaria protection to end users, and manufacturers able to invest in building up quality and distribution expertise.

Looking forward: need for innovation

Looking forward, A to Z faces the prospect of market saturation and a plateau in demand for their bed nets in the 2011 to 2013 time frame. In addition, other African LLIN manufacturing partnerships have recently emerged. Duranet, an LLIN with interim WHO certification, has been manufactured in Uganda on a lowvolume trial basis starting in December 2009, in cooperation with two local companies [16]. AED, Bayer Environmental Science, and Siamdutch Mosquito Netting Company of Thailand developed a system treating finished nets with LLIN chemistry; this system was reportedly installed by Sunflag/Nigeria in 2009 [17]. Sumitomo itself has in 2009 established an Olyset net sewing operation in Ethiopia, in a joint venture with an Ethiopian company with a projected production of 3 million in its first year; Sumitomo has also stated its intention to build a Nigerian factory [18].

One response to these challenges that A to Z is exploring is increased investment in R&D, including adding laboratory facilities and local scientific talent. A to Z has already hired one full time scientist who used to work with WHO. Having benefited from one technologyindeed, having integrated a culture of technology into their businessthey now seek to identify and exploit other opportunities, including potential linkages with domestic scientists and further partnerships with companies like Sumitomo.

In combination with R&D innovation, A to Z faces pressure to continue its business model innovation, through means such as further developing delivery capabilities, and improving packaging and user training. Other innovations may straddle R&D and business, such as cost reduction and more effective bednet efficacy maintenance. While continuous innovation and improvement is a linchpin of business globally, A to Z's experience illustrates the importance of an integrated view of innovation covering technical, business, and social aspects.

A reflection from Anuj Shah illustrates the management's acceptance of both economic and health goals:

…My biggest motivation is the number of lives we are saving. That is a real motivation. That's one. Number two -Africa needs jobs and without people getting jobs, this continent will never develop. The more jobs that are created gives me personal satisfaction that at least people are having an activity, they're earning out of it and they are benefiting out of it. Of course the company is growing so that is really something very, very important for me and really motivates me. Third thingwhen I wake up, it makes me feel that we have to make sure that we have enough orders for the [7000] people to have enough work to carry on, so that itself is a challenge and also to some extent it's a motivation. It always puts you on the edge; we just need to get an order. If you don't have the orders, where are these people going to be sleeping, what are they going to be eating? So that is a real motivation or I can say challenge. I sometimes wish we had more than 24 hours in a day.

Summary

The A to Z story shows that high-quality LLINs can be cost-effectively manufactured in Africa, where malaria is most endemic. This has been achieved without tariffs or other protectionist measures that might lead to significantly higher-priced local ITN production [19]. It demonstrates that health products can be developed by leveraging existing strengths in low-resource settings, leading to health, economic, and technological benefits for these regions. Many such opportunities have been documented in the African context, such as manufacturing common medical supplies [20].

Ultimately, success is enabled by responsiveness to opportunities, willingness to invest and take risks, ability to execute, and strong leadership. A to Z and its partners have not only successfully created an African source for public health goods, but have demonstrated consistent commitment to the endeavor. In the absence of the private enterprise A to Z, it is doubtful that any local public entity would be producing LLINs at the same scale.

Donor funding for bednets played a critical role in creating demand for A to Z's bednets, suggesting that donor and multilateral agencies can kickstart Southern manufacturing by integrating such enterprises into their supply chain. Local funding, scaling up manufacturing, technology transfer, and partnerships with Sumitomo and others all played important roles in A to Z's ascent, as did perceived benefits of local employment and capacity-building; regulatory issues and procurement rules acted as barriers.

Yet continued success is not assuredthe looming plateau in demand for LLINs, competition from imported LLINs sourced from Asian manufacturers, and the recent founding of multiple African sites for LLIN manufacturing all suggest the need for continued innovation to stay in business. In this and other aspects, A to Z is a microcosm of the African-led health innovation enterprise as a whole.

A to Z today: capacity and clinical evidenceThe technology transfer for Olyset bed nets was completed in November 2003, heralding the start of the business partnership between Sumitomo and A to Z Textiles. They began with a trial run of 300 000 LLINS in 2003, reached 1.5 million in 2005, and by 2009 production had grown to 25 million Olyset bed nets per year.
Masum et al. BMC International Health and Human Rights 2010, 10(Suppl 1):S6 http://www.biomedcentral.com/1472-698X/10/S1/S6
AcknowledgementsWe are grateful to Jocalyn Clark, Binesh Haria, Kumar Perampaladas, and Fil Randazzo for comments on drafts of the manuscript, to Hima Batavia for comments and persistent research assistance, to Natasha Bhogal and Jennifer Heys for relevant fieldwork, and to Anuj Shah, Binesh Haria, and other interviewees for their time and willingness to share experiences.Authors' contributions HM, ASD, and PAS conceived the study. RS, ASD, and PAS carried out fieldwork. HM, RS, and KS carried out the analysis. HM, RS, KS, ASD, and PAS drafted the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Published: 13 December 2010
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. Masum, BMC International Health and Human Rights. 101SupplMasum et al. BMC International Health and Human Rights 2010, 10(Suppl 1):S6 http://www.biomedcentral.com/1472-698X/10/S1/S6