Opsonization Assay: Functional Correlate of Protection

Dr. George M. Carlone CDC, Atlanta Elie Metchnikoff

OVERVIEW

• • • • • •

Host protection Serologic markers measured in immunogenicity trials Opsonization – how does it work?

Opsonic assay formats Why it’s important to measure functional activity Correlates we observe in animals & humans

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Host protection against pneumococcal disease is mainly mediated by phagocytosis

•

Phagocytosis – general process describing the engulf ment & destruction of extracellularly-derived materials by phagocytic cells, such as macrophages & neutrophils Opsophagocytosis – opsonophagocytosis is a specific mechanism by which the host protects against infection, with the participation of serum opsonins

• •

Opsonins – antibody and complement (e.g., IgG and C3b) Functional antibody – leads to effective opsonization and recovery from infection

• • • •

Serologic markers that correlate with protection in infants vaccinated with conjugate vaccine IgG ELISA - WHO standardized and validated assay protocol ( www.vaccine.UAB.edu

) Opsonophagocytic assay (OPA) - CDC standard manual killing assay (reference method) protocol ( www.vaccine.UAB.edu

) Antibody avidity – indicator of memory maturation of antibody function & quality Immunological memory immune recall (IgG, rapid, memory T & B cells) ** Evaluated in efficacy study

Comparison of serologic markers that correlate with protection in infants and/or adults Laboratory Assays Conjugate Vaccine Poly. Vaccine Infant Adult Infant Adult IgG ELISA OPA Avidity Memory Animal Model passive protect.

Cellular Immunity cytokines, etc.

X X X X X X X X ?

X?

X ?

na na na na na na X X NO NO X X

Opsonization - how does it work?

antigen IgG cell swells and bursts first complement proteins complement cascade complement inserts into cell wall

IgG and C3b are known as opsonins and the process of attachment is called opsonization * subclasses differ in C’ deposition capacity Receptor for IgG Receptor for iC3b (high & low avidity) Phagocytic cell www.cat.cc.md.us/.../ opsonization/u1fig26n.html

Opsonophagocytosis of pneumococci requires bacteria, Ab, C’, and phagocytic cells With Ab and C’ Without Ab and C’ Pnc are not being engulfed Pnc are being engulfed & killed

www.lsumc.edu/campus/micr/opson.htm

Engulfment and killing

1. attachment of bacteria / beads phagocyte 2. engulfment phagosome 4. respiratory burst stimulation of NADPH oxidase phagolysosome residual body lysosomes 3. degranulation: fusion of granules to phagosome bacterial killing and digestion www.cvm.uiuc.edu/.../ vp331/Intracell_Bacteria

Capsules are anti-phagocytic

(interferes with attraction of phagocyte, engulfment and recognition of cell as foreign) * The thicker the capsule the more Ab required for OPA www.uic.edu/.../ slide0231.htm

Opsonic Assay Formats

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Manual assays (viable cells) – measures killing CDC reference method (Romero-Steiner), multiplexed, antibiotic resistant (Kim, Nahm; Bogaert) radiometric (Jonsdottir), other

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Flow cytometric (non-viable cells) – measures uptake single color (Martinez; Jansen) three color multiplex (Martinez) bead based target multiplexed (Martinez), other

Why is functional (OPA) activity important to measure for vaccine evaluation?

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Opsonophagocytic activity correlates with protection, however, Ab conc. may not always corr with function

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Formal efficacy trials will likely not be done Serotypes in new vaccine formulations will not proven efficacy have

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Vaccines from different manufacturers will likely be different (structurally, immunologically, etc.) Therefore, functional activity is an essential measurement for vaccine comparison

What do animal models tell us about potential correlates of protection?

Protective activity of serotype 6B specific IgG against bacteremia after challenge with a 6B isolate IgG 48hr after immunization 0.0 ug/ml 0.05

0.1

0.2

Absence of bacteremia 48hr after challenge Serum 89SF #/total/(%) 2/37 (5.4) Clinical sample #/total/(%) 5/55 (9.1) P value 0.7

6/16 (38) 23/29 (79) 17/17 (100) 19/35 (54) 24/24 (100) 9/9 (100) 0.4

0.03

NA

Johnson, et al. 1999. JId. 180:133.

Correlation between bacteremia and OPA titer in a mouse passive protection model

` 6 5 4 3 2 1

1:8 Serotype 1 Serotype 4 Serotype 5 Serotype 6B Serotype 18C Serotype 23

0 -1 -2 -3 -4 -5 -6

75%

n = 731 r = .84

P

< .001

0 20 40 60 80 100

Infant mice nonbacteremic Infant mice nonbacteremic at 48 hr (%)

Johnson, et al. 1999. JId. 180:133.

What do human trials tell us about potential correlates of protection?

11-Valent Conjugate Vaccine Response in Filipino Infants Serotype 4 Serotype 6B 18 wk old 10 mo old + EIA & OPA pre (6 wk) EIA Serotype 14 EIA Serotype 19F Regression …. 18 wk old ___10 mo old EIA Puumalainen, et al. 2003. JID. 187:1704. EIA

Reverse cumulative distributions of post dose 3 ELISA Ab for 7 serotypes in infants (Black, et al., North Calif. Trial) 100 80 60 40

97.9

VE = 1 - (1-.979) (1-.129) = .976

20

12.9

0 0.01

0.1

1 10 100 Ab Concentration

Ignoring Ab levels in controls obtains [Ab] prot = .20 µg/ml

Vax Control

Correlation of ELISA and OPA (North CA KP infant study) Jodar, et al. 2003. Vaccine Total N = 79

10000 R = 0.80

( p < 0.0001) 7VPnC Control

1000 R = 0.80

( p < 0.0001) 100

1:8

10

0.2ug/ml

1 0.01

0.1

1 10

ELISA Concentration (



g/ml) ELISA concentration of 0.20



OPA titer of 1:8

SUMMARY

• • • • • •

OPA is a correlate Animal & human Immunogenicity of protection OPA protection data appear to agree ELISA & OPA are primary end-points (good correlation) OPA formats include killing (std.) and uptake Functional activity is influenced by avidity may be influenced assays and age by study design and/or vaccine formulation, dose, scheduling, etc.

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Formal efficacy trials will likely not be done