Smallpox: Residual Antibody after Vaccination

Of all the microorganisms and toxins, poxviruses (Orthopoxvirus)have the greatest potential for use by terrorists. These virusescan spread rapidly through the environment following initialinfection. In 1980, the World Health Organization EradicationProgram discontinued vaccination for smallpox and declared thatthe disease had been eliminated. With the threat of smallpoxvirus as a bioterrorism weapon, questions have been asked aboutthe persistence of protection (as offered by antibodies) followingvaccination with vaccinia virus vaccine. To address this, serafrom 204 adults vaccinated as children were tested by enzymeimmunoassay (EIA) for the presence of vaccinia virus antibody.Of the 204 individuals whose sera were examined for the presenceof vaccinia antibody, 165 (80.9%) had been vaccinated once and39 (19.1%) had been vaccinated at least twice. Of the 165 serafrom individuals vaccinated once, 112 (67.9%) were positive.Of the 39 sera from individuals vaccinated more than once, 31(79.5%) were positive. The presence of a vaccination scar atthe time of blood collection was not determined. Fifty-six nonvaccinatedindividuals, under 30 years of age, were tested by EIA; fourof these (7.1%) were positive for vaccinia virus antibody byEIA. Forty-four EIA-positive and 16 EIA-negative sera were alsotested by serum neutralization (SN) as a comparison with theEIA test results; one serum (negative by EIA) was SN positive.No attempt was made to ascertain any demographics other thanage (date of birth) and "remembered" times of vaccination.

INTRODUCTION

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Introduction

In November 2001, United States citizens became acutely awareof the potential for bioterrorism within our borders. With theintroduction of anthrax bacilli (Bacillus anthracis) into theU.S. Postal Service system, the threat of other forms of bioterrorismbecame a reality. Biological terrorism, or bioterrorism, involvesthe use or the threat of using disease-spreading microorganismsand/or toxins as weapons of mass destruction. The use of B.anthracis as a weapon of terrorism has resulted in a heightenedreview of other potential bioterrorism agents. Smallpox virus(variola virus) is considered to pose the greatest risk.

In 1980 the World Health Organization Smallpox Eradication Programdeclared that the disease, and the need for a vaccine with possibleadverse reactions, had been eliminated (1-3). However, stocksof variola virus were "officially" maintained in the UnitedStates and in the Soviet Union for experimental purposes (6).The dissolution of the Soviet Union led to concerns regardingsafety control of a number of State properties, including stocksof variola virus. Other unknown illicit smallpox virus sourcesmay exist, and these may serve as a potential for bioterrorismthreats.

Most adults 35 years old and older were vaccinated for smallpoxat least once prior to entering school. An unknown parameterof this smallpox vaccination, however, is the duration of itsprotection, as determined by the presence of antibody. It isgenerally accepted that the original vaccination would be protectivefor about 5 to 10 years, although the World Health OrganizationCommittee on International Quarantine suggested that internationaltravelers be vaccinated within the 3 years prior to travel (5).Multiple vaccinations provide long-term protection (4); however,the duration of antibody from the single original smallpox vaccinationhas not been recently evaluated. Do adults who received a singlevaccination as a child still have any residual antibody? Manyindividuals are concerned with regard to protection againstsmallpox infection and specifically with whether they themselveshave antibodies. Each serum donor was questioned regarding thenumber of times he or she was vaccinated.

In this study, the presence of humoral antibody in vaccinatedand unvaccinated (control) individuals was investigated. Thosewho had received additional booster vaccinations were noted,and their results were reported separately from those for individualswho had received a single vaccination. As it is not known whetherantibody detected by enzyme immunoassay (EIA) is protective,it was of interest to determine if another antibody test wouldyield similar results. Accordingly, for comparison, 60 of theEIA-tested sera were also examined by serum neutralization (SN).

MATERIALS AND METHODS

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Materials and Methods

Two hundred four sera from adults of various ages, but at least35 years of age, were submitted to the ESOTERIX Infectious DiseaseCenter laboratory by their physicians for the detection of vacciniavirus antibody. All claimed to have been vaccinated as children.In the final scoring, results for individuals with more thanone vaccination were tabulated separately from those for thegroup that had been vaccinated only once. In the tabulationof subjects, there were only two criteria: vaccinated once orvaccinated more than once. As there were 39 sera available fromindividuals vaccinated more than once, these were also testedby EIA. The laboratory received only serum samples; the presenceor absence of vaccination scars was not reported.

After blood collection by venipuncture, sera were separatedfrom the clotted blood by centrifugation and then refrigeratedat 2 to 8°C. If not tested within 1 week of collection,the sera were frozen at -20°C. Serum titers were not determined,as the purpose of this study was to ascertain the number ofantibody-positive responses resulting from vaccination.

Approximately half the sera were collected in California, andthe other half were collected in Texas. Laboratory personnelperforming EIA and SN tests were unaware of the individual'svaccination status. The majority of test individuals were unableto recall precisely when they received their primary vaccinationbut thought it was prior to starting kindergarten or elementaryschool. Fifty-six sera from individuals less than 30 years ofage and knowingly never vaccinated were similarly tested byEIA as negative controls.

Vaccinia virus (American Type Culture Collection, VR-156), derivedfrom the New York City Department of Health IHD strain, wasgrown and maintained in this laboratory in Vero cells. Followinggrowth, virus preparations were frozen and thawed three timesand then centrifuged at 1,500 rpm in a Sorvall refrigeratedultracentrifuge to remove cell debris. The clarified preparationwas again centrifuged at 20,000 rpm for 2 h, and the virus pelletwas suspended in 2.0 ml of phosphate-buffered saline (PBS),pH 7.2. This test antigen was then titrated by EIA against seriallydiluted positive serum in a box titration as described below.

Serially diluted antigen (100 µl/well) was added to a96-well polystyrene EIA plate and incubated for 1 h at roomtemperature. The plate was then washed three times with PBScontaining 0.1% Tween 20 (PBST) and once with PBS. Nonspecificantigen sites were blocked with PBS containing 3% bovine serumalbumin (BSA) for 1 h at room temperature. The plate was againwashed with PBST and once with PBS. Serially diluted antibody-positivecontrol serum (100 µl) was inoculated into duplicate wellscontaining the serially diluted antigen. Two wells of each antigendilution were inoculated per serum dilution. The EIA plate wasthen incubated for 1 h before each well was washed three timeswith PBST and once with PBS.

A 1:1,000 dilution of anti-human immunoglobulin G-alkaline phosphataseconjugate (100 µl; Kirkegaard & Perry, Gaithersburg,Md.) was added to each well, and the plate was incubated atroom temperature for 1 h. The conjugate dilution was determinedby titrating against a serum-antigen box titration in triplicate.The wells were washed three times in PBST and then once withPBS. Paranitrophenyl phosphate substrate (100 µl/well;Kirkegaard & Perry), prepared 10 min prior to use, was addedto each well, and the plate was incubated for 15 min at roomtemperature in the dark. The reaction was stopped with 100 µlof 5% EDTA per well, and results were read at 405 nm with a630-nm reference filter. The optimum antigen titer and serumdilution were determined to be 1:100. Positive and negativecontrol sera, PBS, and antigen-only wells were included witheach test serum.

Based on the results of the box titration, test sera were screenedat a 1:100 dilution in PBS containing 3% BSA. The sera weretested in duplicate by using 100 µl of a 1:100 dilutionper well of a 1:100 antigen dilution. Concerns that BSA containedcowpox antibody were minimized by the control tests, which containedBSA and were negative.

In addition to the EIA, as a comparison, 60 sera that had beentested by EIA (44 EIA positive and 16 EIA negative) were alsoexamined for SN antibody. After the virus titer in Vero cells(10^6.0/0.1 ml) had been determined, 100 50% tissue culture infectivedoses of virus was added to a serum dilution of 1:5 in PBS.The virus-serum mixtures were incubated at 37°C for 1 h.Two Vero cell monolayer wells in a 24-well culture plate wereinoculated with 0.2 ml of each virus-serum mixture. Test andcontrol (positive and negative) cultures were read at 24 and48 h.

RESULTS

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Results

Two hundred four sera were tested by EIA; of these, 165 (80.9%)were from individuals that had been vaccinated once and 39 (19.1%)were from individuals who had been vaccinated more than once.Of the 165 sera from individuals who had been vaccinated once,112 (67.9%) had vaccinia antibody at the test dilution of 1:100and 53 (32.1%) were negative. Of 39 sera from subjects thathad been vaccinated more than once, 31 (79.5%) were positive.Although the primary purpose of testing these sera was to ascertainif antibody persisted long term after a single vaccination,the results of multiple vaccinations are also reported. Sixty-eightpercent of the single-vaccine recipients' sera were positivefor antibody, compared to 79.5% of the sera from subjects withmultiple vaccinations.

When the data are arranged according to the decade of birth(Table 1), it is apparent that most of the sera were collectedfrom individuals with birth dates between 1921 and 1970. Ineach of these decades, a significant number of positive resultswere obtained. However, the percent positive in individualsborn in the decades from 1921 to 1940 (89.2%) is somewhat greaterthan that in individuals born in 1941 to 1970 (65.2%). Bothindividuals born prior to the 1920s were vaccinated more thanonce, as was the one born between 1971 and 1980.

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TABLE 1. EIA results reported according to decade of vaccinated individuals' birth^a

Of the 60 sera (44 were EIA positive and 16 were EIA negative)tested by SN, 59 (98.3%) were negative. The one SN-positiveserum was from an individual born in 1948 and was negative byEIA.

Fifty-six sera from individuals under 30 years of age and presumablynever vaccinated were also tested by EIA (control group). Toour knowledge, none of the individuals were in the Armed Forces.In addition, they did not remember a previous smallpox vaccination.Of these 56 samples, 52 (92.9%) were negative and 4 (7.1%) werepositive by EIA.

DISCUSSION

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Discussion

Two hundred four sera were tested for vaccinia virus antibody.Of these, 165 (80.9%) sera were from individuals who had beenvaccinated once and 39 (19.1%) were from individuals who hadbeen vaccinated at least twice. One hundred forty-three (70.1%)sera retained some evidence of vaccinia virus antibody as determinedby EIA. Sixty-one (29.9%) sera were negative, including thosefrom 8 of 39 individuals vaccinated at least twice. Of the 143EIA-positive sera, 112 were from individuals (78.3%) vaccinatedonly once and 31 (21.7%) were from individuals vaccinated atleast twice.

While the intent of the study was to determine the number ofindividuals who still had antibody after a single vaccination,there were 39 sera from individuals vaccinated more than once.It was of interest, therefore, to ascertain if there was anydifference in residual antibody between single and multiplevaccinations. Interestingly, 67.9% of the 165 individuals vaccinatedonce had antibody, while 79.5% (31 of 39) of those who receivedmultiple vaccinations were antibody positive. This suggeststhat multiple vaccinations may provide longer-lasting immunityagainst smallpox infection.

Inasmuch as the intent of the study was to determine the presenceor absence of vaccinia virus antibody, the sera were screenedat a single dilution without determining endpoint titers.

The data presented for the number of positives per decade ofage show that the greatest percentage of EIA positives was amongthe number of donors who were born in 1921 to 1970. Consideringthat the number of sera collected from individuals with earlieror later birth dates was not significantly different, it isnot clear why there are more positives among individuals bornbetween 1921 and 1940 than among those born in 1941 to 1970.This may be a reflection of the number of sera tested in eachdecade or perhaps chance or coincidence.

The issue of smallpox protection is moot and would require additionalstudy. If antibody detectable by EIA is protective, these resultswould suggest that individuals with some humoral antibody 30to 35 years after vaccination might be protected against a bioterrorismattack with smallpox virus. It was of interest, therefore, tocompare vaccinia virus antibody detection by EIA with detectionby another serological test, the SN test. Of the 60 sera testedfor SN antibody, the only SN-positive sample was a single EIA-negativeserum. The failure to demonstrate SN antibody in the EIA-positivesera brings into question the protective capability of antibodydetected by EIA. One may also ask why the one serum negativeby EIA is SN positive. Additional testing with other immunologicalmethods would be required to determine what constitutes protection.

It is highly probable, inasmuch as the members of the genusOrthopoxvirus are all closely related antigenically, that ifthere is protection against vaccinia virus, there is protectionagainst the other members of the genus Orthopoxvirus (7, 8).Jezek et al. (7) indicate that smallpox vaccination protectsagainst monkeypox. Smallpox virus is only one of the poxvirusesconsidered in terms of bioterrorism: poxviruses other than variolaand vaccinia (cowpox) viruses, including camelpox virus, monkeypoxvirus, fowlpox virus, and others, are recognized by the biologicalcommunity (8).

Perhaps a smallpox attack would precipitate a protective anamnesticresponse or reveal a reduction in disease severity in EIA-positiveindividuals. If this did occur, then any smallpox outbreak inthe population would be predominantly in the younger (nonvaccinated)age groups. The presence of antibody, as detected by EIA, ina large segment of the population may indicate that there ismore protection to smallpox than originally thought.

The EIA control group of 56 individuals with no known historyof vaccination indicated that positives do occur. Of the 56sera, 52 (92.9%) were EIA negative. The four (7.1%) EIA-positiveindividuals may have been unknowingly vaccinated. As mentionedabove, many of the tested subjects stated they were vaccinated,but the exact dates were vague. It may be that there were anumber of individuals who have been vaccinated but are unawareof the event.

The results presented here indicate the need to explore whatimmune response, by EIA, SN, or other tests, is a true indicationof protection from infection by the smallpox virus. The presenceof a cell-mediated immune response to vaccinia virus in individualsvaccinated before 1980 should be studied in order to evaluatethe results reported here. Cell-mediated immunity may reflectthe true persistence of immunity to smallpox infection. In retrospect,the identification of vaccination scars at the time of phlebotomymight have been useful.

ACKNOWLEDGMENTS

We thank Vernon R. Benson, I Med, and Charles A. Coltman, CancerTherapy and Research Center, for their support in providingthe sera. The assistance of Sara D. Reyna, Toni L. Sparrow,and Mark S. Diamond is greatly appreciated.

FOOTNOTES

* Corresponding author. Mailing address: ESOTERIX Infectious Disease Center, 7540 Louis Pasteur, San Antonio, TX 78229. Phone: (210) 614-7350. Fax: (210) 614-7355. E-mail: sy.kalter{at}esoterix.com.

REFERENCES

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