Modifying the vaginal microbiome to protect against HIV Laurel Lagenaur Guest Researcher NIH HIV Infection in Women Around the Globe • Need for HIV prevention strategies for women • In 2013, almost 60% of all new HIV infections occurred among women, particularly young women and adolescent girls aged 15–24. Women Age 24 – 50% HIV+ Men Age 24 – 6% HIV+ HIV is Transmitted Across Mucosal Surfaces • HIV infection in women occurs in the epithelium of the vagina and cervix • Infection of underlying target cells (mostly CD4+ T cells) From Tom Hope Mucosal Surfaces are a Living Community • All mucosal surfaces are continuously exposed to a community of microorganisms Lactobacilli • Vagina and Cervix Vaginal Lactobacillus sp. are Epithelial Cell found in 51-90 % women H2O2-Colonization • L. crispatus, L. jensenii, D-lactic acid producers L. gasseri (and L. iners) L-lactic acid Vaginal Microbiota is Relevant to Human Health Healthy Vagina Dysbiosis Bacterial Diversity H2O2-producing Lactobacillus Low Diversity High Diversity Lactobacillus = Defense against Increased risk of infections (BV, rUTI) urogenital pathogens Increased risk of preterm birth, other Contribute to low vaginal pH OB/GYN complications Produce antibacterial substances Increased inflammation and risk of HIV Block pathogen binding, i.e. competitive exclusion Cervicovaginal Bacteria are a Major Modulator of the Host Inflammatory Responses • A recent paper by Anahtar et al. (senior authors Walker, Fichorova, Kwon, Immunity 2015 studied a cohort of South African women • A majority of these women had low abundance of Lactobacillus • Low Lactobacillus abundance together with high ecological diversity strongly correlated with genital pro- inflammatory cytokine concentration Four community types • Community types – Group 1, L. crispatus, very low alpha diversity – Group 2 L. iners low diversity – Group 3 Gardnerella moderate diversity – Group 4 Prevotella high diversity Anahtar et al. Immunity 2015 Women with CT4 had high inflammatory cytokines – Increases in IL-1α, IL-1β, TNF-α, IFN-γ, IL-10 and IL-8 IL-1α IL-1β TNF-α IFN-γ IL-10 IL-8 Immunity 2015 Osel’s Technology Modulating the Vaginal Microbiome to Prevent HIV Modulating the Vaginal Microbiome to Prevent HIV • Engineer vaginal Lactobacilli to produce an HIV entry inhibitor (recombinant live biotherapeutic) HIV • Colonize the mucosa modified Lactobacillus jensenii 1153 HIV inhibitor (H2O2) producer Lactobacillus - Natural defense, lactic acid, “anti- inflammatory” HIV - Genetically modified to produce HIV inhibitor an HIV entry inhibitor - Protection at the site of HIV Lactobacillus infection Strain selection process • Screened >20 isolates of L. crispatus, L. jensenii and L. gasseri from healthy women • Selected L. jensenii 1153 - Although variations of the microbiota exist within individuals over time and in different geographical settings, L. jensenii is found in 17%-41% of women across many regions of the world Strain selection • Selected L. jensenii 1153 L. jensenii 1153 - Growth rate-doubling time - D-Lactic acid production - Adherence to tissue - Activity against Gardnerella and Staphylococcus - Transform (no endogenous plasmids) • Broad Institute/J. Craig Venter Institute - sequenced and annotated genome 1153 Selecting an inhibitor; HIV is a diverse virus B A B C C G AE AG B D A C A C B HG C Global distribution of HIV-1 subtypes and recombinants. Trends in Molecular Medicine, Volume 18, Issue 3, 2012, 182 - 192 HIV Inhibitor Selection • Tested expression multiple HIV entry inhibitors in Lactobacillus • Selected HIV inhibitor protein Cyanovirin-N Inhibitor Selection Cyanovirin-N (NCI) - Identified by National Cancer Institute drug screen (Cyanobacterium) - Activity against all subtypes - Concentration of CV-N produced by Lactobacillus [272 nm] - Inhibited >85% HIV viruses in a representative panel of major subtypes - CV-N active pH 4-8 (vaginal(vaginal pHpH range)range) HIV Inhibitor Selection •Cyanovirin-N Binding Courtesy of Carole Bewley Ultrastructural: Cell+ HIVBaL + Lactobacillus HIV HIV cell Identified optimal Lactobacillus strain, strong HIV inhibitor that worked against multiple subtypes and at an appropriate pH range Next we constructed a recombinant Lactobacillus and tested it for fitness Constructing a recombinant • Stably integrated mCV-N Integration plasmid gene into the Lactobacillus chromosome by orfA` mCVN orfA` homologous recombination orfA L. jensenii 1153 chromosome • Biologically active level of inhibitor expression using a Integrate into chromosome constitutive ribosomal promoter Secretion of mCVN Liu et al. 2006, AAC Fitness and Phenotypic Characteristics of L. jensenii 1153 with Integrated mCV-N Recombinant strains produced equivalent amounts of D-Lactic acid as the parent (WT) 4.5 4 D-Lactic acid 3.5 3 WT1153 wt 2.5 R1pox1 2 1.5 R2pepO 1 D-LacticAcid (g/L) Lactic Acid (g/L) Acid Lactic - 0.5 D 0 0 1 OD600 2 3 OD600 Fitness and Phenotypic Characteristics of L. jensenii 1153 with Integrated mCV-N Recombinant strains have similar growth rates to the parent (WT) 20 18 ) 8 16 14 12 WT1153 wt ) 8 10 growth rates R2pepO 8 R1pox1 6 4 Cell Numbers (10 2 Cell numbers (10 numbers Cell 0 0 200 400 600 800 TimeTime Minutes (Minutes) Phenotypic Characteristics of L. jensenii 1153 with Integrated mCV-N Recombinant strains secreted biologically active mCV-N 1153 WT Rec 1 Rec 2 OD600 0.5 1.0 3 0.5 1.0 3 0.5 1.0 3 kDa 14 mCV-N 11kDa 6 Recombinant strain produced H2O2 We had constructed a fit recombinant L. jensenii expressing mCV-N Next we wanted to test it in vivo Macaque model Intravaginal Inoculation of L. jensenii producing mCV-N in Macaques In initial studies….. Pellet + 3% hydroxyethyl cellulose Lactobacilli Vaginal Epithelial Cell Freshly prepared Rhesus macaque overnight culture Brown and Hopps Modified Gram stain Recovery of L. jensenii-mCV-N from Rhesus Macaques following 5-day dose Followed 10macaques10 for 6 weeks 9 10 Vaginal colonization 108 107 recovered 106 105 104 103 102 Lactobacillus jensenii 101 Lactobacillus CFU recovered Lactobacillus 100 7 14 21 28 35 42 49 Days PI L. jensenii-mCV-N We showed that our L. jensenii recombinant could colonize the macaque Next we wanted to prove that it was making mCV-N protein in situ Proof of Principle • Colonized rhesus macaques Lj 1153-1666 Lj 1153-2666 • Cervical vaginal lavage M1 M2 M3 M4 M5 M6 taken weekly Week 0 (before inoculation) Day 1 • mCV-N protein (24 hrs after inoculation) detected by immunoblot for at least 6 weeks Week 3 Week 6 Proof of Principle • Colonized rhesus macaques • Immunohistochemistry on vaginal biopsies for mCV-N protein Pre-immune Anti-CV-N • mCV-N protein localized vaginal lumen on mucosal surface at the site of virus entry mucosal surface 1:4000 1:4000 We showed that our L. jensenii recombinant could colonize the macaque and make mCV-N protein in situ Now we wanted to challenge macaques with Simian (S)HIV to test whether infection could be prevented SHIV Repeated Dose Challenge • Two arm study – 12 macaques received L. jensenii expressing mCV-N – 12 control (8 macaques received HEC placebo gel/4 no gel – Macaques were challenged each week for 6 weeks with (S)HIV – Monitored for viral infection each week • Repeated dose challenges measure the rate of infection per exposure • Infection rate about 33% macaques/exposure (100- 1000X higher than amount found in human) • Monitor time to infection Repeated Dose Challenge-Power Analysis Control group: n=12 Experimental group: n = 12 31 challenges 61 challenges 11 infected animals 8 infected animal 35.4% infection rate 13.1% infection rate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SHIV Repeated Dose Challenge Results • Controls: 35% infection per exposure • LB-mCV-N: 13% infection per exposure • 63% reduction in acquisition p<0.004 • 6X reduction in viral load (macaques with breakthrough infection) 61 challenge 8 infections=13.1% 31 challenges/11 infected=35.4% Lagenaur et al. 2011 Mucosal Immunology Efficacy in a SHIV challenge model Now we moved forward with formulation of a product that could be used in the “real world” Formulation into a Product-MucoCept • Formulation Prototype tablet -Developed a vaginal ~100 mg disintegrating tablet 10 9 -Tablets are potent 5X1011 8 CFU/gm and stable (1yr) at 7 M 6 4oC or 25oC 5 4 Limit of detection by culture - Colonize macaques 3 L. jensenii 1153-1666 (CFU/swab) L. jensenii 1153-1666 Detection by PCR 2 10 1 Log Tablet administration 0 7 14 21 Days Post Dosing Lagenaur et al. 2015 PLoSOne SHIV challenge Days 0 7 10 14 17 21 24 MucoCept Tablet Placebo Tablet 34715 Survival proportions: Davis Study Part 1 all macaques 35065 100 36230 Active VDT 36262 infected 39332 50 Percent survival VDT placebo 34103 (P) infected 0 37712 (P) infected 0 1 2 3 Challenges 38603 (P) infected Snapshot during SHIV challenge SHIV challenge Days 0 7 10 14 17 21 24 MucoCept Tablet Placebo Tablet Protected Phylum:Firmicutes;Bacilli 34715 MucoCept Placebo Order: Family: Genus Tablet 100%# Tablet Turicibacterales;#Turicibacteraceae;#Turicibacter# 100%# Lactobacillales;#Streptococcaceae;#Streptococcus#Turicibacterales;#Turicibacteraceae;#Turicibacter# Lactobacillales;#Streptococcaceae;#Lactococcus#Lactobacillales;#Streptococcaceae;#Streptococcus# Lactobacillales;#Lactobacillaceae;#Lactobacillus#Lactobacillales;#Streptococcaceae;#Lactococcus# 75%# Lactobacillales;#Enterococcaceae;#Enterococcus#Lactobacillales;#Lactobacillaceae;#Lactobacillus#