Interaction with other flaviviruses (pre-existing immunity, co-infection, cross-reactivity)

Alan D.T. Barrett Department of Pathology

Sealy Center for Vaccine Development

University of Texas Medical Branch

Galveston TX

Flavivirus genome

50nm particle.

SS, +RNA genome.

10 genes, 3 structural.

Beck, A. Barrett, ADT. (2015) Exp Rev Vaccines. 1-14.

2

Flavivirus E protein epitopes

• Studies with human and mouse polyclonal sera show extensive serologic

cross-reactivities between flaviviruses in terms of physical (ELISA) and

biological (HAI and neutralization) assays

• Studies with mouse, non-human primate, and human monoclonal

antibodies show essentially the same result that all flaviviruses studied to

date have a range of E protein epitopes ranging in flavivirus cross-reactive

(e.g., mab 4G2 or 6B6C-1), to flavivirus intermediate (e.g., mab 1B7), to

serocomplex specific (e.g., DENV-1 to DENV-4; mab MDVP-55A), to

flavivirus species specific (e.g., mab 3H5 that is DENV-2 specific). Strain

specific epitopes are rare.

• Flavivirus infection induces a range of antibodies, including those that

recognize multiple flaviviruses. A second, but different, flavivirus infection

potentiates induction of flavivirus cross-reactive antibodies.

• Most epitopes are “conformational” or “quaternary”. Very few epitopes are

linear. Very few epitopes appear to elicit high titer neutralizing antibodies.

RH: Rabbit hyperimune sera Gould et al., 1985

Reactivity of anti-E protein mouse monoclonal antibodies raised

against YF 17D vaccine with YF and 37 other flaviviruses

RH: Rabbit hyperimune sera Gould et al., 1985

Reactivity of anti-E protein mouse monoclonal antibodies raised

against YF 17D vaccine with YF and 37 other flaviviruses

Reactivity of anti-E and anti-NS1 protein mouse monoclonal

antibodies raised against YF 17D vaccine with different YF strains

Flavivirus NS1 protein

Less flavivirus cross-reactive epitopes than E protein, but some still

identified.

NS1 has been studied as a vaccine “immunogen” but does not induce

neutralizing antibodies only complement fixing antibodies (antibody

dependent cellular cytotoxicity).

NS1 protein is a nonstructural protein that is secreted from cells

during flavivirus infection. Is used as a diagnostic tool for a dengue

infection but is it flavivirus-species specific?

Antibodies to NS1 and NS5 can be used as diagnostic markers of a

flavivirus species-specific infection. Could be very useful for

evaluation of inactivated or subunit vaccines.

Reactivity of anti-NS1 protein mouse monoclonal antibodies raised

against YF 17D vaccine with YF and 37 other flaviviruses

RH: Rabbit hyperimune sera Gould et al., 1985

Reactivity of anti-NS1 protein mouse monoclonal antibodies raised

against YF 17D vaccine with YF and 37 other flaviviruses

RH: Rabbit hyperimune sera Gould et al., 1985

Complexities of evaluating flavivirus immune responses

• Flavivirus serology is a “minefield”. Hard to

serologically identify an infection as due to a

particular flavivirus unless the individual is

flavivirus-naïve.

• Karl Johnson called flaviviruses the “Hall of

Mirrors”

• Challenging to assess and interpret

immunological data due to cross-reactivity.

How do you do a flavivirus neutralization test? • De ja vue!

• Cell type, virus strain, and assay type will influence results.

• Plaque reduction neutralization test (PRNT)

PRNT50?

PRNT60?

PRNT80?

PRNT90?

• Micro-neut

• Flow cytometry

• A standardized, validated assay will be important to quantitate

neutralizing antibodies

Zika cross-reactivity – flavivirus naive

Lanciotti et al., 2008

Lanciotti et al., 2008

Zika cross-reactivity – Zika as a secondary flavivirus infection

Yap Island results and flavivirus vaccines

• If ZIKV is the secondary flavivirus infection, the individual may not

have the highest neutralizing antibody titer to ZIKV as compared to

other flaviviruses.

• The neutralizing antibody titer may be higher against a previous

(primary?) flavivirus infection rather than the most recent

heterologous flavivirus (secondary?) infection.

• Individuals who have been previously vaccinated against yellow

fever, Japanese encephalitis, (candidate) dengue, or tick-borne

encephalitis can have a rapid and high neutralizing antibody titer

following infection by a heterologous flavivirus. Usually, seen with

mosquito-borne flavivirus infections (JE, YF and DEN vaccine).

Vaccination in the context of pre-existing flavivirus immunity

Vaccine Efficacy, According to Serotype and Age Group.

Hadinegoro SR et al. N Engl J Med 2015;373:1195-1206

Complexities of serologic diagnostics • IgM and neutralizing antibody testing can identify recent ZIKV

infections if the individual is flavivirus-naïve.

• ZIKV (ELISA and PRNT) antibody test results can be difficult to

interpret; particular in secondary flavivius infections (i.e., infection

by a different flavirus to that of the primary infection) because of

serologic cross-reactivity with other flaviviruses.

• Similarly, it is difficult to identify the particular flavivirus infection

when the individual has had a previous flavivirus vaccination (YF

or JE [or DEN?]).

• IgM positive is a presumptive recent flavivirus infection but may

not be flavivirus species specific if the individual has had a

previous flavivirus infection or vaccination. i.e., ZIKV IgM positive

may not be indicative of a recent ZIKV infection.

Conclusions

We have a large “tool box” to measure flavivirus immune responses.

There are approximately 70 flaviviruses, many of which have

extensive serologic cross-reactivity.

Interpretation of flavivirus serology results is complex. This may

make evaluation of some vaccine candidates difficult.

Clinical evaluation of inactivated or subunit vaccines may have

advantages in using serologic evaluation of nonstructural proteins.