Mucosal protection to HIV-1: what to induce and how to measure it? AIDS Vaccine 07, Seattle, August 20-23, 2007 Robin Shattock St George’s, University of London A number of critical questions remain unanswered • The relative role of cell free vs. infected cells in mucosal transmission, • The potential mechanism of viral transport across mucosal surfaces, • The identity, frequency, location and role of the primary targets, • The relative importance of these by mucosal route, • The relative impact of mucosal responses on these different pathways. Langerhans Cells in Ectocervix HIV can be found associated with Langerhans cells located in the interstitial space of the squamous epithelium T. Hope CD1a (Langerhans cells) GFP-VPR labeled HIV after photoactivation Cell free or cell associated virus? Does innate immunity modulate mucosal HIV transmission? • Innate immune responses occur within minutes or hours of pathogen entry • Innate antiviral factors (intracellular and secreted) modulate viral replication • Their role in modulating transmission/susceptibility is not fully understood • The innate responses to viral exposure may be critical in influencing primary and secondary adaptive immune response • TLRs and non-TLR pattern recognition receptors modulating the strength, quality and persistence of adaptive immune responses. Can innate immunity control viral replication in mucosal tissue? 1e4 1e4 1000 1000 Drug/Small-molecule Medium Only (control) 1000 1e4 Drug/Small-molecule pIpC (control) 1000 1e4 What humoral responses needed at the time of infection What needs to be measured and how? • Are neutralizing antibodies the only responses contributing to robust mucosal protection? • Do other functional characteristics have equal or additional importance? • How is this modulated by antibody isotype? • What is the role of locally produced antibodies? • How much spill over of systemic antibodies is there into mucosal compartments? • Is this changed by sexual arousal? • Are luminal antibodies important? Are local events important for mucosal protection? •Passive transfer of b12 Mab requires 25mg/kg for sterile protection against vaginal challenge. - Parren…Burton et al. J Virol, 2001. 75:8340-8347. •Greater protection was achieved against vaginal vs. IV challenge - Mascola et al. Nat Med, 2000. 6:207-10 •Topically applied b12 Mab (5mg) provides 80% protection (4/5) - Veazey et al. Nat Med, 2003, 9:343-346 •IgA secreting cells in the iliac lymph nodes of TILN immunized macaques correlate significantly with protection from infection - Lehner et al. Nat Med. 1996, 2:1054-5. What are the correlates of protective antibody responses? •neutralizing antibodies? •IgG, sIgA, IgM? •Systemic vs local? •Kinetics (when and where)? 2.5 2.5 2.0 1.5 COS 01 COS 02 Cos 03 Cos 04 Cos 05 Cos 06 0.5 90 Standard PEP014 Cervical os PEP014 Vaginal vault PEP014 COS / VV-unspiked 2.0 1.5 1.0 0.0 101 102 103 1 / dilution 104 105 80 IgA 70 IgG 60 IgM-Penta 50 40 30 20 10 0.5 0.0 Mass Difference (Hz) 3.0 1.0 100 IgA 3.0 Absorbance (450nm) Absorbance (450nm) IgG 2F5 Binding Differentials of HIV-Bal 101 102 103 1 / dilution 104 105 0 1.E-05 1.E-04 1.E-03 1.E-02 HIV Relative Dillution Factor 1.E-01 1.E+00 Greater emphasis on development of functional mucosal antibody assays. Lights ŅONÓ LUC Cγ2 Infection C γ2 C γ3 γ3 C Tzm-bl cell Pseudovirus Blockage of Transcytosis Neutralization assays – cellfree infection TZM-Bl, PBMC & M0 Cell-cell transmission Defining predictive Mucosal assays Moog - P10-1 & 16 OA05-03 Mucosal explants Mucosal explants (polarized) - transmission - replication and neutralization of ‘acute’ env / HIV http://www.altaweb.it/europrise/neutnet.html 2F5 IgA provides the most potent inhibition of HIV-1 dissemination from cervical by migratory DCs HIV Mucosa Increased virus replication Afferent and spread lymphatics Draining lymphoid tissue HIV-1 (R5) Polystyrene Ring d4 d7 d11 60 50 IgG 40 IgA IgM 30 Nylon Membrane 20 10 0 IgG 50 10 2 0.4 IgA 50 10 2 0.4 IgM 50 antibody conc (ug/ml) 10 2 0.4 neg pos Can protective effector immune responses be maintained at the portal of entry? • Inefficient targeting of mucosal antigen presenting cells (APCs). • Rapid leakage from the administered sites. • Degradation of antigens by enzymes in mucosal secretions • Differences in immune response during the menstrual cycle. However, appropriate cervicovaginal delivery of antigens can induce better local IgA responses than either rectal or nasal delivery Vaginal immunization can induce localized and systemic immune responses Dominant isotypes IgG >< IgA Hormonal regulation +++ Inductive site – to + Effector site ++ Contribution from +++ the circulation (~ 50%) •Vaginal immunization induces Cholera vaccine-specific cervical IgA Ab responses after vaginal immunization during the midfollicular menstrual cycle phase. - Kozlowski et al Journal of Immunology, 2002, 169: 566–574. •The magnitudes of these responses are 20-fold for IgA antitoxin and 7.1-fold for IgG antitoxin - Wassen et al, Clinical Vaccine Immunology 2006, 13, 202-207 GMP product Stability data of GP140 final product after 12 months 12 Months Test Day 0 ≤ -55 °C 2-8 °C 25 ± 2 °C OD (µg/ml) 530 528 522 569 SEC (% framents and monomer) <5 <5 <5 <5 SDS-PAGE pass pass pass pass Native PAGE pass pass pass pass WB pass pass pass pass Osmolality (mOsmol/kg) 313 311 313 320 pH 7.4 7.3 7.3 7.3 Visual appearance colourless, clear colourless, clear colourless, clear colourless, clear Native PAGE stained with Coomassie Elution profiles of size exclusion chromatography 12 months -55 °C 12 months 25 °C 12 months 2-8C 70 60 50 50 50 30 mAU [214nm] 60 40 40 30 40 20 20 10 10 10 15 20 25 time [min] 30 35 40 monomers 0 0 10 trimers 30 20 0 12 months 25C 70 60 mAU [214nm] mAU [214nm] 12 m onths 2-8 °C 12 months -55C 70 10 15 20 25 30 35 40 10 15 time [min] Antigen very stable, unlikely to require refrigeration 20 25 time [min] 30 35 40 Can vaginal immunization induce specific immune responses? Intravaginal application of 100μg (620μl) ZM96 gp140 in gel formulation 01 3 5 8 10 12 15 17 19 20 ZM96GP140-specific Serum IgG Titres D20 Titre ZM96gp140-specific Mucosal IgG Titres D49 Titre Vagina April 25000 Vestibule April 70 60 20000 15000 IgG Titre IgG Titre 49 10000 50 40 30 20 5000 10 0 0 21 22 23 24 25 26 Rabbit number 27 28 29 30 21 22 23 24 25 26 27 28 29 Rabbit number Cranage et al P13-74 30 Nanoparticles are internalised by DC and incorporated into endolysosomes 100000 * * 10000 1000 particles lysosomes gp140 + Particle-A gp140 + particle-B gp140 + particle-C gp140 alone gp140 + alum Loxley et al P13-44 Yellow: particles within lysosomes Lacey et al AIDS Vaccine 07, Seattle, August 20-23, 2007 EMERGING ISSUES ON …… Improving Defences at the Portal of Entry (Summary of the recommendations from an Enterprise Working Group)