RSV Vaccine History, Animal Models, and Assays€¦ · RSV Vaccine History, Animal ... March 23-24, 2015. Barney Graham, MD, PhD. Vaccine Research Center. NIAID, NIH. Dale and

RSV Vaccine History, Animal Models, and Assays

WHO Consultation on RSV Vaccine DevelopmentMarch 23-24, 2015

Barney Graham, MD, PhDVaccine Research CenterNIAID, NIH

Dale and Betty Bumpers

Vaccine Research CenterNational Institute of Allergy and Infectious DiseasesNational Institutes of Health

Outline

• Review of RSV-induced pathology• History of FI-RSV vaccine-enhanced disease• What can animal models tell us? • What can assays tell us?• Reinterpretation of FI-RSV antigenicity

RSV BronchiolitisCiliated bronchiolar epithelium

Type 1 alveolar pneumocyte

Johnson JE, Graham BS, et al. Modern Pathology 2007; 20:108-19.

3

RSV causes airway obstruction

Epithelial damage Lymphoid aggregatesMucus

Obstructed airway

Johnson JE, Gonzales RA, Olson SJ, Wright PF, Graham. The histopathology of fatal untreated human respiratory syncytial virus infection. Modern Pathology 2007; 20:108-19.

BBBAAA

Polarized ciliated epitheliumBronchiolitis

Presenter

Presentation Notes

RSV infects ciliated cells in the small airways. Although usually referred to as a bronchiolits, RSV also infects the alveolar epithelium. This is an immunostain positive Type 1 alveolar pneumocyte. Infected Type 2 pneumocytes and infected macrophages can also be seen. Signs of lower airway disease (for example hypoxia) are commonly found in persons infected with RSV even without lower respiratory tract symptoms, so alveolitis should be considered a normal component of RSV pathology.

RSV in Immunosuppressed Child

Courtesy of John DeVincenzo

Neutrophils are the dominant cell in BAL, while mononuclear cells are most common in tissue

aa

Presenter

Presentation Notes

Relative segregation of mononuclear cells and polymorphonuclear leukocytes in airway. Peribronchiolar mononuclear inflammatory infiltrate is primarily submuscular, and is peripheral to the smooth muscle (arrow). 'L' indicates the airway lumen in the 1931 case (a). While neutrophils are commonly found in BAL, the predominant cells in the peribronchiolar tissues are mononuclear. Neutrophils can sometimes be found migrating across gaps in the muscularis (arrow) toward the airway lumen and interspersed within the epithelium (dashed arrows) in 1946 case (b). Original magnification 250 (a and b).

History of FI-RSV Vaccine Enhanced Disease in Clinical Trials

<1966 – Live and inactivated RSV given parenterallywithout benefit

1966-7 – 4 independent studies using Pfizer lot 100 formalin-inactivated RSV did not protect and caused enhanced disease

>1967 - Live RSV IM, live-attenuated RSV IN given without harmSubunit F (F+G+M, FG, F+G) and G (BBG2Na) given to adults and children with pre-existing immunity (2-3 fold rise in NT; >10-20 fold rise in ELISA titers)

Pfizer Lot 100 FI-RSV• Bernett strain of RSV isolated in primary human embryonic

kidney from throat swab at NIH, May 1961 and passaged X3• Passaged X10 and produced in vervet monkey kidney cells• 3 early (day 4) and 11 late (day 7) sublot harvests combined• Clarified by Millipore SM filtration• 72 hours at 36°C with formalin 1/4000 wt/vol• 50,000 rpm pellet and 25-fold concentration in EMEM in

Earle’s salt solution• Alum precipitation (4 mg/ml) (?aluminum potassium sulfate)• Storage at 4°C with polymyxin, neomycin, streptomycin, and

benzethonium preservatives• IM delivery on a 0,1 or 0,1,2 or 0,1,4 month schedule• Control was Parainfluenza I then trivalent Para 1,2,3

Chemistry of aldehyde fixation

Formaldehyde - Cross-links free amino groups with –CH2- or causes an amino alkylation of a carbonyl group resulting in a β-amino-carbonyl compound.

Glutaraldehyde – can react amine, thiol, phenol, and imidazole functional groups on amino acids, but preferentially reacts with ε-amino groups to cross-link lysine residues

Paraformaldehyde – fixation similar to formaldehyde

Papers reporting FI-RSV vaccine-enhanced disease (Am J Epidemiol 1969)

• Chin et al. (0,1 schedule; 4 mo. – 10 year; 43 <1 yr.)– Eosinophilia– CF response <6 mo (36%), 6-18 mo (55%), 2-9 yrs. (84%)– Higher rate and severity of illness in vaccinees (?higher virus load)

• Fulginiti et al. (0,1,2 schedule; 6 mo.-7 yrs.)– 7/51 (13.7%) vaccinees vs. 1/65 (1.5%) TPV recipients hospitalized– Of 19 with >4X CF responses only 9 had NT responses

• Kapikian et al. (0,1,2 schedule; 5-65 mo. of age; only 2 <1yr.)– 9/13 vaccinees with pneumonia vs. 4/47 nonvaccinated (5 & 0 hosp.)– Relatively high CF antibody and NT response, and no eosinophilia

• Kim et al. (0,1,4 schedule; 2-7 mo. of age)– Peribronchial monocytic infiltrate with excess of eosinophils– Relatively high CF antibody response in vaccinees– High lymphoproliferation index in vaccinees

FI-RSV Vaccine-Enhanced Disease

Kim et al. Am J Epidemiol 1969;89:422

Vaccine n Infected (%) Hospitalized (%)* Deaths**FI- RSV 31 20 (65) 16 (80) 2FI-PIV-1 40 21 (53) 1 (5) 0

N % 4-fold rise Mean fold rise

Post RSV mean fold rise

NT with C’ 23 43 2.6 21 (12 of 16)

CF 23 91 30 165 (15 of 16)*

* 2 received 1 injection, 8 received 2 injectionsNoted 21/54 infected in 1962/3 trial and 10/21 required hospitalization

** 14 and 16 mo. of age, 3 injections starting at 2 and 5 mo. of age, one had no CF or NT response, the other had CF=128 and NT=48. both had bacterial pneumonia complicating RSV

* Paraflu recipients after natural infection mean fold rise = 71 (13 of 14)

11

Presenter

Presentation Notes

Reinfection is common. Up to 50% of children infected in the 1st year of life are reinfected in the 2nd year of life, and all of us are reinfected every two to three years throughout our lives. These images illustrate the fact that a persons 1st infection, while not conferring preventive immunity, does induce a response that diminishes illness from a subsequent infection.

Histopathology in FI-RSV Vaccine-Enhanced Disease

FI-RSV Immunization Results in a Discordance Between Functional and Binding Antibody

Murphy et al JCM 1986; 24:197 & 1988; 26:1595

VaccinationNatural Infection

Pre Post Pre Post

ELISA

Fusion Inhibition

Animal Models of RSV

• Rodents– Mice

• immunocompromised• Pneumovirus of mice

– Cotton rats– Guinea pigs

• Large animal– Bovine– Ovine

• Nonhuman primates– Chimpanzees– African Green monkeys– Baboons

• Other– Ferret– Chinchilla

Murine Model of RSV

Determinants of FI-RSV Vaccine Enhanced Illness in BALB/c Mice

Johnson & Graham et al. J Virol 2004; 78:6024

% E

osin

ophi

ls in

BA

L

05

1015202530354045

FI-Vero FI-rRSVwt

FI-rRSV∆G

FI-rRSVGep-

FI-r RSV∆SH Johnson et al.

J Virol 2004; 78:8753

Gob-5 Muc5ac Mucus by PAS Alveolitis

Presenter

Presentation Notes

Our initial motivation for developing a murine model for RSV was to understand the pathogenesis of the FI-RSV vaccine-enhanced disease. This slide demonstrates the principle feature of the immune response to FI-RSV. Lung extracts from mice challenged with RSV after FI-RSV immunization show a dominant IL-4 mRNA pattern whereas the dominant response in mice immunized with live RSV given IM or IN is IFN-gamma. This is the basis for the current hypotheses involving Th2-mediated disease in RSV infection.

Potential Advantages of Cotton Rats Over Mice

• 3d old cotton rats >1 log10 pfu more replication in nose than adults with delayed viral clearance

• More prominent neutrophil infiltrate• Slightly more permissive than mice, particularly

neonates

RSV Replication in Cotton Rat Lung and Nose

NoseLung

Prince et al. Am J Pathol 1978; 93:771

FI-RSV Enhanced Disease Endpoint in Cotton Rats

Prince GA, et al. J Virol 1986; 57:721

Problems with Rodent Models

Mice• Semi-permissive• Relatively high body temperature• Type I IFN not inhibited by NS1/2• Large inoculum directly to lung• BALB/c model characteristics

based largely on two unusual T cells – a Vβ13.2 M282-90 CD8 and a Vβ14 G184-198 CD4

Cotton Rats• Semi-permissive• Large inoculum directly to lung• Does not exhibit illness• Limited immunological assessment

based on inbred strains and reagents

• Difficult to distinguish neutrophils from eosinophils

Caveats – 1) cannot assess histopathology in absence of viral replication, 2) cannot reinfect after primary infection

Bovine model of bRSV

Geraldine Taylor. Current Topics in Microbiology and Immunology 2013

Bronchiolitis

Lung consolidation

Alveolitis

Infection of bronchiolar epithelium

Caveats – 1) bRSV is not hRSV, 2) generally small group sizes in each experiment, 3) limited immunological reagents

Immune Response Patterns Associated with FI-RSV Vaccine-Enhanced Illness

Polack et al. J Exp Med 2002; 196:859 Graham et al. JI 1993;151:2032

CD4+ Th2-biased response↓

Allergic inflammation

Antibodies with poor NT activity ↓

Immune complex deposition

22

Presenter

Presentation Notes

Our initial motivation for developing a murine model for RSV was to understand the pathogenesis of the FI-RSV vaccine-enhanced disease. This slide demonstrates the principle feature of the immune response to FI-RSV. Lung extracts from mice challenged with RSV after FI-RSV immunization show a dominant IL-4 mRNA pattern whereas the dominant response in mice immunized with live RSV given IM or IN is IFN-gamma. This is the basis for the current hypotheses involving Th2-mediated disease in RSV infection.

Class I Fusion Glycoproteins

Viral membrane Viral membrane Viral membrane Viral membraneViral membrane Viral membrane

PIV F Influenza HA HIV-1 gp160 Ebola GP CoV SRSV F

** HR2 TMFP HR1

F1F2

69 212 322 333 343 358 367 382 393 416 422 439 550

Regions of high mobility

5743721

70 116 126 50027

3131

Antigenic sites ∅ III V∅V IVII I

RSV F mediates viral entry

24

100 nm

16 n

m11

nm

Post-fusion inactive form of F

F on the viral envelopeRSV budding from a cell

Prefusion native form of F

Location and Conformations of F

Liljeroos L et al. PNAS 2013;110:11133-11138 25

RSV buds as a filamentous particle from cholesterol-rich lipid raft microdomains

RSV F conformation changes as virus morphology evolves

Liljeroos L et al. PNAS 2013;110:11133-11138

10 nm

27

Presenter

Presentation Notes

Tomography of A2 HRSV virions. Virion morphology ranges from completely filamentous (A) to completely spherical (C) with intermediate forms (B and D) that have some tubularly curved parts but are otherwise spherically curved. Spherical particles are highly deformable when in the proximity of other particles and the membrane proximal to the neighboring particle is free of glycoprotein spikes (E). Virions in A–E are illustrated schematically in F in alphabetical order. Black arrows: side views of the RNP; white arrows: top views of the RNP; green arrows: secondary density layer under the membrane in a spherical particle. (Scale bar, 100 nm.) Tomographic slices are 3.8-nm-thick.

RSV morphology and protein content

Prefusion F has unique sites of vulnerability to neutralizing antibodies

Conclusions

• FI-RSV caused enhanced illness in children <24 mo. old• Antibody response showed a much greater fold increase in

complement-fixing than neutralizing or fusion-inhibiting activity (i.e. high ratio of binding to functional antibody)

• Animal models and original studies suggest a Th2-biased immune response was associated with enhanced disease

• Most NT-sensitive epitopes are on pre-F conformation• Heating virus at 37°C for 72 hours causes F to flip into post-F

conformation

NIAID Vaccine Research Center

Peter Kwong, Jason McLellan, Gordon Joyce, Guillaume Stewart-Jones, et al.

Viral Pathogenesis Laboratory Structural Biology Section

Sung-Han Kim, Syed Moin, Barney Graham, Kaitlyn Morabito, Azad Kumar, Kevin Graepel, Kayvon

Modjarrad, Man Chen, Tracy Ruckwardt, Allison Malloy, Jason McLellan, Jie Liu, Erez Bar-Haim,

Joan Ngwuta, April Killikelly

Special thanks to Peter Collins31

Documents

RSV Vaccine History, Animal Models, and Assays€¦ · RSV Vaccine History, Animal ... March 23-24, 2015. Barney Graham, MD, PhD. Vaccine Research Center. NIAID, NIH. Dale and