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Cholera, a diarrheal disease primarily affecting vulnerable populations in developing countries, is estimated to cause disease in more than 2.5 million people and kill almost 100,000 annually. An oral cholera vaccine (OCV) has been available globally since 2001; the demand for this vaccine from affected countries has however been very low, due to various factors including vaccine price and mode of administration. The low demand for the vaccine and limited commercial incentives to invest in research and development of vaccines for developing country markets has kept the global supply of OCVs down. Since 1999, the International Vaccine Institute has been committed to make safe, effective and affordable OCVs accessible. Through a variety of partnerships with collaborators in Sweden, Vietnam, India and South Korea, and with public and private funding, IVI facilitated development and production of two affordable and WHO-prequalified OCVs and together with other stakeholders accelerated the introduction of these vaccines for the global public-sector market.

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Vaccinating a buffer of individuals around a case (ring vaccination) has the potential to target those who are at highest risk of infection, reducing the number of doses needed to control a disease. We explored the potential vaccine effectiveness (VE) of oral cholera vaccines (OCVs) for such a strategy. This analysis uses existing data from a cluster-randomized clinical trial in which OCV or placebo was given to 71,900 participants in Kolkata, India, from 27 July to 10 September 2006. Cholera surveillance was then conducted on 144,106 individuals living in the study area, including trial participants, for 5 y following vaccination. First, we explored the risk of cholera among contacts of cholera patients, and, second, we measured VE among individuals living within 25 m of cholera cases between 8 and 28 d after onset of the index case. For the first analysis, individuals living around each index case identified during the 5-y period were assembled using a ring to define cohorts of individuals exposed to cholera index cases. An index control without cholera was randomly selected for each index case from the same population, matched by age group, and individuals living around each index control were assembled using a ring to define cohorts not exposed to cholera cases. Cholera attack rates among the exposed and non-exposed cohorts were compared using different distances from the index case/control to define the rings and different time frames to define the period at risk. For the VE analysis, the exposed cohorts were further stratified according to the level of vaccine coverage into high and low coverage strata. Overall VE was assessed by comparing the attack rates between high and low vaccine coverage strata irrespective of individuals' vaccination status, and indirect VE was assessed by comparing the attack rates among unvaccinated members between high and low vaccine coverage strata. Cholera risk among the cohort exposed to cholera cases was 5-11 times higher than that among the cohort not exposed to cholera cases. The risk gradually diminished with an increase in distance and time. The overall and indirect VE measured between 8 and 28 d after exposure to a cholera index case during the first 2 y was 91% (95% CI 62%-98%) and 93% (95% CI 44%-99%), respectively. VE persisted for 5 y after vaccination and was similar whether the index case was a young child (<5 y) or was older. Of note, this study was a reanalysis of a cholera vaccine trial that used two doses; thus, a limitation of the study relates to the assumption that a single dose, if administered quickly, will induce a similar level of total and indirect protection over the short term as did two doses. These findings suggest that high-level protection can be achieved if individuals living close to cholera cases are living in a high coverage ring. Since this was an observational study including participants who had received two doses of vaccine (or placebo) in the clinical trial, further studies are needed to determine whether a ring vaccination strategy, in which vaccine is given quickly to those living close to a case, is feasible and effective. ClinicalTrials.gov NCT00289224.

•Bridging study demonstrating the equivalence of two variations of Euvichol®.•The 600L thimerosal-free Euvichol® is safe and immunogenic in adults and children.•The scale-up of Euvichol® allows expanding global access to oral cholera vaccine. To contribute to the global demand for oral cholera vaccine (OCV), the production of Euvichol® was scaled up with elimination of thimerosal. To demonstrate the equivalence of the variations, a study was carried out in the Philippines. Healthy male and female adults and children in Manila were randomized to receive two doses of Euvichol® two weeks apart from either the 100L (Comparator) or the 600L (Test) variation. Primary and secondary immunogenicity endpoints were respectively geometric mean titer (GMT) of vibriocidal antibodies (two weeks post second dose) and seroconversion rate (two weeks after each dose) against O1 Inaba, Ogawa, and O139 serogroups. The GMT of vibriocidal antibodies against O1 Inaba, Ogawa, and O139 two weeks post first dose was also measured. To show the equivalence of two variations of Euvichol®, the ratio of GMT and the difference of seroconversion rate between Test and Comparator vaccines were tested with equivalence margin of [0.5, 2.0] for GMT ratio and of 15% for seroconversion rate, respectively. Safety assessment included solicited reactogenicity within 6 days after each dose and unsolicited and serious adverse events. A total of 442 participants were enrolled. For the overall population, equivalence between Test and Comparator was demonstrated for vibriocidal antibody response against O1 Inaba and Ogawa serotypes and O139 serogroup in both modified intention-to-treat (mITT) and per protocol analysis, since the 95% confidence intervals (CI) of GMT to any serotypes were within the lower and upper boundary [0.5, 2.0]. Seroconversion rates after two doses also showed equivalence for O1 Inaba, Ogawa, and O139. The vaccine was safe and well tolerated, similarly between the two groups. The study results support the equivalence of the 600L Euvichol® to the 100L formulation in healthy children and adults. The 600L Euvichol® is safe and immunogenic in adults and children. ClinicalTrials.gov registration number: NCT02502331.

Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.

Combination treatments with agents that inhibit protease and reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) decrease mortality and slow disease progression. 1 The development of resistance to these drugs, however, limits the benefit of such treatments. 2 , 3 There have been reports of the transmission of HIV-1 variants that are resistant to nucleoside and non-nucleoside inhibitors of reverse transcriptase. 4 – 9 The transmission of HIV-1 variants that are resistant to protease inhibitors could represent an important emerging clinical and public health problem. We report a case of transmission of an HIV-1 variant with multiple mutations that conferred resistance to both protease . . .

Membrane traffic critically regulates most aspects of neuronal function. Neurons express many neuronal-specific proteins that regulate membrane traffic, including the poorly understood small transmembrane proteins neural-specific gene 1 and 2 (Nsg1/NEEP21 and Nsg2/P19). Nsg1 has been implicated in regulating endosomal recycling and sorting of several important neuronal receptors. Nsg2 is largely unstudied. At steady-state, Nsg1 and Nsg2 only partially co-localize with known endosomal compartments, and it was suggested that they mark a neuronal-specific endosome. Since Nsg1 localizes to and functions in the dendritic endosome, we set out to discover how Nsg1 and Nsg2 localization to endosomes is regulated in primary rat hippocampal neurons, using quadruple immunolocalization against endogenous proteins, live imaging of dendritic endosomes, and interference approaches against the endosomal regulators Rab5 and Rab7. In contrast to previous conclusions, we now show that Nsg1 and Nsg2 are not resident endosomal proteins, but traffic rapidly from the cell surface to lysosomes and have a half-life of less than two hours. Their partial co-localization with canonical endosomal markers thus reflects their rapid flux towards degradation rather than specific targeting to a singular compartment. These findings will require rethinking of how this class of endosomal proteins regulates trafficking of much longer-lived receptors.

Background CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS. Methods The capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses. Results The predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region. Conclusions CABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.

The human β-chemokine receptor CCR5 is an important cofactor for entry of human immunodeficiency virus-type 1 (HIV-1). The murine form of CCR5, despite its 82 percent identity to the human form, was not functional as an HIV-1 coreceptor. HIV-1 entry function could be reconstituted by fusion of various individual elements derived from the extracellular region of human CCR5 onto murine CCR5. Analysis of chimeras containing elements from human CCR5 and human CCR2B suggested that a complex structure rather than single contact sites is responsible for facilitation of viral entry. Further, certain chimeras lacking the domains necessary to signal in response to their natural chemokine ligands retained vigorous HIV-1 coreceptor activity.

Killed whole-cell oral cholera vaccines (kOCVs) are becoming a standard cholera control and prevention tool. However, vaccine efficacy and direct effectiveness estimates have varied, with differences in study design, location, follow-up duration, and vaccine composition posing challenges for public health decision making. We did a systematic review and meta-analysis to generate average estimates of kOCV efficacy and direct effectiveness from the available literature. For this systematic review and meta-analysis, we searched PubMed, Embase, Scopus, and the Cochrane Review Library on July 9, 2016, and ISI Web of Science on July 11, 2016, for randomised controlled trials and observational studies that reported estimates of direct protection against medically attended confirmed cholera conferred by kOCVs. We included studies published on any date in English, Spanish, French, or Chinese. We extracted from the published reports the primary efficacy and effectiveness estimates from each study and also estimates according to number of vaccine doses, duration, and age group. The main study outcome was average efficacy and direct effectiveness of two kOCV doses, which we estimated with random-effect models. This study is registered with PROSPERO, number CRD42016048232. Seven trials (with 695 patients with cholera) and six observational studies (217 patients with cholera) met the inclusion criteria, with an average two-dose efficacy of 58% (95% CI 42–69, I2=58%) and effectiveness of 76% (62–85, I2=0). Average two-dose efficacy in children younger than 5 years (30% [95% CI 15–42], I2=0%) was lower than in those 5 years or older (64% [58–70], I2=0%; p<0·0001). Two-dose efficacy estimates of kOCV were similar during the first 2 years after vaccination, with estimates of 56% (95% CI 42–66, I2=45%) in the first year and 59% (49–67, I2=0) in the second year. The efficacy reduced to 39% (13 to 57, I2=48%) in the third year, and 26% (−46 to 63, I2=74%) in the fourth year. Two kOCV doses provide protection against cholera for at least 3 years. One kOCV dose provides at least short-term protection, which has important implications for outbreak management. kOCVs are effective tools for cholera control. The Bill & Melinda Gates Foundation.