Studying Cell Immune Responses to a Live Flu Vaccine in Healthy Adults

Two types of influenza vaccines are currently licensed in the US, trivalent inactivated
vaccine (TIV), administered by intramuscular injection, and LAIV, administered by nasal
spray. Both vaccines are safe and effective in their approved age groups. Neutralizing
antibody in the serum has been found to be a correlate of protection for TIV, but the immune
correlates of protection for LAIV are not known. Defining the origin and nature of
transcriptional responses to LAIV in URT in infected and bystander epithelial and lymphocyte
cells in healthy adults will be a highly informative first step in a systems approach toward
understanding the molecular basis of viral replication restriction and the regulation of the
local mucosal immune responses following LAIV administration.

This natural history study will use a systems biology approach to identify LAIV replication
niches among a variety of URT cell types and characterize the host immune response to LAIV.
Healthy volunteers aged 18-49 years will be prescreened for low (<1:10) serum HAI titer
against the component influenza vaccine virus strains (influenza A H1N1 and H3N2, and
influenza B) of the licensed seasonal LAIV. Ten HAIlow or -negative individuals will be
vaccinated intranasally with LAIV (n=8) or will receive saline intranasally (n=2). One week
prior to and 2 days after vaccine administration, an NP specimen will be collected using
flocked NP swabs. A blood sample will be collected at the time of NP swabbing and on Days 7
and 28 after vaccination. Total subject participation time from enrollment/baseline to the
final study visit will be 5 weeks.

We propose to recover cells from NP swab samples and sort individual cells of different
subsets based on specific surface phenotypic markers. We will then utilize
microfluidics-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) to
quantify transcripts from bulk and single cells. These transcripts will include
strand-specific influenza RNA for determining virus replication, genes for assigning cells to
specific epithelial or lymphocyte subpopulations, selected genes in the IFN signaling
pathways to determine innate immune responses, and genes involved in activation and effector
functions of different immune cell subsets. Results will be analyzed with several
bioinformatics tools, with an emphasis on the differential signaling responses between
various cells types. The mucosal transcriptional data will be correlated with B and T cell
immunity markers and traditional serology (HAI and neutralization assays) before and after
vaccination to identify key factors affecting the immune response to LAIV.

- INCLUSION CRITERIA:

- Adult males and females between 18 and 49 years of age.

- Females of child-bearing potential must have a negative serum beta-human
choriogonadotropin (HCG) on Day -7 and agree to use an effective birth control method
for the duration of the study (for example, pharmacologic contraceptives including
oral, parenteral, and transcutaneous delivery; condoms with spermicide; diaphragm with
spermicide; intrauterine device; abstinence from heterosexual intercourse, surgical
sterilization). All female subjects will be considered being of child-bearing
potential except those who have undergone hysterectomy and those in whom menopause
occurred at least 1 year prior to the study.

- General good health, without significant medical illness, physical examination
findings, or significant laboratory abnormalities as determined by the investigator.

- Agree to storage of blood, NP swab, and RNA specimens for future research.

- Available for the duration of the study. Subjects must be willing and able to make
follow-up visits as specified by the protocol.

EXCLUSION CRITERIA:

- Any contraindiciations for adults for the receipt of LAIV:

--Hypersensitivity to eggs, egg proteins, gentamicin, gelatin, or arginine,or
life-threatening reactions to previous influenza vaccination.

- Currently breast-feeding.

- Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic,
rheumatologic, autoimmune, or renal disease by history, physical examination, and/or
laboratory studies including urine testing. Clinically significant alanine
aminotransferase (ALT) levels, as determined by one of the protocol investigators will
be exclusionary at baseline, prior to vaccination.

- Evidence of upper respiratory tract illness (URI) including fever, sore throat, and
rhinorrhea. Enrollment of subjects with evidence of URI on Study Day -7 will be
temporarily deferred.

- Deviated nasal septum or nasal obstruction.

- Body Mass Index greater than 35.

- NIH employee involved in direct patient care, consistent with the NIH CC policy that
employees should avoid patient care for 5 days following receipt of LAIV. In addition,
NIH employees must not be under the supervision of the study principal or associate
investigators.

- Behavioral or cognitive impairment or psychiatric disease that in the opinion of the
investigator affects the ability of the subject to understand and cooperate with the
study protocol.

- Seropositive to the influenza A component viruses of seasonal LAIV (serum HAI titer of
greater than or equal to 1:10).

- Has had medical, occupational, or family problems as a result of alcohol or illicit
drug use during the past 12 months.

- Other condition that in the opinion of the investigator would jeopardize the safety or
rights of a subject participating in the study or would render the subject unable to
comply with the protocol.

- History of anaphylaxis.

- Current diagnosis of asthma or reactive airway disease (within the past 2 years).

- History of Guillain-Barr(SqrRoot)(Copyright) Syndrome.

- Positive enzyme-linked immunosorbent assay (ELISA) and confirmatory Western blot tests
for human immunodeficiency virus-1 (HIV-1).

- Positive ELISA and confirmatory test (e.g., recombinant immunoblotassay) for hepatitis
C virus (HCV).

- Positive hepatitis B virus surface antigen (HBsAg) by ELISA.

- Known immunodeficiency syndrome.

- Use of oral, injected, or inhaled corticosteroids (excluding topical preparations) or
immunosuppressive drugs within 30 days prior to enrollment in the study.

- Use of any herbal supplement 30 days prior to study enrollment.

- Receipt of 2012-2013 influenza vaccine (TIV or LAIV).

- Receipt of a live vaccine within 4 weeks or a killed vaccine within 2 weeks prior to
enrollment in the study.

- History of a surgical splenectomy.

- Receipt of blood or blood-derived products (including immunoglobulin) within 6 months
prior to study vaccination.

- Current smoker.

- Travel to the Southern Hemisphere 30 days prior to study enrollment or anticipated
travel to the Southern Hemisphere during the study.

- Travel on a cruise ship 30 days prior to study enrollment or anticipated travel on a
cruise ship during the study.

- Receipt of another investigational vaccine or drug within 30 days prior to enrollment
in the study.