How to determine protective immunity in the post-vaccine era

abstract

The ability to determine an individual's susceptibility to infection relies heavily on the assay used, and the ability to correlate results of the assay to a clinical interpretation. Current rubella immunity screening methods identify total rubella IgG antibodies circulating in the serum, however both humoral and cell mediated immune responses have been shown to contribute to protection from infection. Therefore, antibody screening assays may under-estimate immunity in some populations. In fact, waning antibody titers over time in a large prenatal population were recently documented in North America, and the trend has been echoed in other countries that have achieved elimination through universal rubella vaccination. Despite decreasing antibody titers, the number of acute rubella cases has not increased in these populations, suggesting that the lower antibody levels may still be protective. Based on the changing epidemiology in universally vaccinated populations, it may be time to reassess the level of antibody that indicates immunity to rubella infection.

Keywords:

• prenatal immunity testing
• rubella antibody levels
• rubella cut off
• rubella immunity
• waning immunity

The role of cell mediated and humoral immune responses in vaccination

The success of the rubella vaccine is in part due to its ability to elicit both a cell mediated and a humoral immune response. The use of a live-attenuated virus in the vaccine closely mimics interactions that would be observed between the host and a wild type virus. The RA27/3 virus (currently used for vaccination in Canada and the US) is known to replicate within host cells, similar to wild type virus, and can be detected in the blood of volunteers following vaccination.¹ The presence of cervical lymphadenopathy following vaccination in some individuals further suggests vaccine derived viral replication within host cells.² RA27/3 is well documented to induce a strong antibody response in vaccinated individuals, and the presence of rubella IgG antibodies has been observed years to decades after initial vaccination.^3,4 Likewise, T cell responses have been shown to be long-lived following vaccination. In fact, lymphocyte proliferation was observed in T cells exposed to rubella-specific peptides 14–16 years after a single dose of RA27/3.^5,6 Additionally, T cell proliferation has been shown for peptides known to elicit a neutralizing antibody response, suggesting a cognate T helper and B cell interaction may occur following vaccination.⁶ Clinically, individuals with T cell deficiencies or other cellular immunity abnormalities (such as leukemia), who have high levels of rubella specific antibodies, have developed rubella disease following exposure to wild type virus.⁷ Together, these results suggest full vaccine effectiveness is dependent on an individual mounting both an antibody and a cell-mediated immune response.

Choosing the appropriate test and assay cut off

The greatest challenges in assessing and individual's susceptibility to infection are (1) to identify an appropriate test to assess immunity, and (2) to determine a cut off which would represent protection from infection. The monitoring of cell-mediated immune levels in response to a viral antigen in the laboratory is a highly labor intensive procedure, typically involving radioactive elements, and is performed only in specialized laboratories. In contrast, the detection of circulating antibodies can be performed relatively easily using high throughput serological assays (often a chemiluminescent microparticle immunoassay (CMIA)). Thus, the level of rubella IgG antibody is used as a surrogate marker for protection. In 1985, the Rubella Subcommittee of the National Committee on Clinical Laboratory Standards (NCCLS) set a level of >15 IU/ml for rubella IgG antibodies as the indicator of immunity.⁸ In light of further epidemiological investigations, and additional studies indicating that individuals with low levels of antibody (<15 IU/ml) produced a secondary immune response upon vaccine challenge rather than a primary immune response,^1,9-11 these cut offs were revised by the Subcommittee from 15 IU/ml to 10 IU/ml in 1992.¹² However, since 1992, the rubella cutoffs have not been assessed. Recent publications have shown that college students who received rubella vaccination during childhood, but who had low, or no detectable antibody response, mounted a secondary immune response upon challenge with rubella vaccination.¹³ Additionally, gamma interferon release following exposure to attenuated rubella virus, was detectable in all individuals with low antibody levels, and was not statistically different from those with high antibody titers,¹³ suggesting low antibody levels may not always be indicative of susceptibility to infection.

In countries where rubella vaccination was incorporated into universal childhood vaccination schedules, the level of rubella IgG has been declining over time.^3,4,14 The numbers of acute rubella and congenital rubella syndrome (CRS) cases however have not increased despite waning antibody levels in the population,^4,15,16 highlighting the effectiveness of the current vaccine programs. In the context of herd immunity, the lower antibody levels may therefore be sufficient to protect an individual from rubella infection. The observed decline over time may be due to the increased proportion of women who rely only on vaccine-induced immunity. There are 2 prevailing theories as to why antibody titers are declining: firstly, the vaccine may not produce the same level of immune activation as wild type infection. In a small cohort study, individuals with natural immunity had higher levels of antibody compared to women with vaccine-induced immunity,¹³ suggesting that the vaccine-induced immune response may not be as long lasting as the response from a wild type virus. Therefore, in universally vaccinated populations, as more of the population base relies on vaccine-induced immunity, the levels of antibody will decline as the cohort ages. The second theory suggests that as a result of universal vaccination, the amount of circulating virus will decline in a population, and may be eliminated (barring external re-introduction of the virus, such as travel related cases). The loss of circulating virus in a population therefore may prevent adequate immune system “boosting” to maintain antibodies at protective levels, which may naturally occur when wild type virus is circulating in a population. Regardless of the root cause of waning antibody titers, the epidemiology of universally vaccinated populations is changing. In fact, many countries, including Finland (>4 IU/ml seronegative, 4–7 IU/ml equivocal) and Australia (>4 IU/ml seronegative, 4–15 IU/ml equivocal), are already using lower cutoffs for population screening.^4,16 Latent class modeling of a large prenatal patient population in Canada showed that rubella antibody levels <2–5 IU/ml may more accurately identify those women who lack immunity to infection in universally vaccinated populations.³ These results are consistent with a recent study that compared 8 different serological assays against the immunoblot (Mikrogen Diagnostik, Neuried, Germany), which uses whole viral lysate to identify rubella specific antibodies. All immunoblot negative samples corresponded to rubella antibody levels <2 IU/ml on the Abbott Architect platform,¹⁷ suggesting that levels >2 IU/ml may indicate immunity to infection. However, a more accurate measure of immunity is through detection of neutralizing antibodies, rather than total rubella IgG antibody levels. As all of the currently available commercial screening assays detect total rubella IgG levels, and do not detect the level of neutralizing antibody (used to determine the recommended cutoff of 10 IU/ml),¹² the level of true protective antibody levels may not be accurately measured by commercial screening assays. Significant variability in precision and reproducibility across manufacture's platforms was observed when identical samples were tested on 5 different assays,¹⁸ suggesting that a single cut off may not be able to be extrapolated to all rubella IgG screening assays. Additionally, while total IgG assays correlate well with high rubella IgG levels, the reproducibility of low rubella IgG titers, and particularly those around the cutoffs, is poor.¹⁸ Large scale studies examining the level of neutralizing antibody in vaccinated populations who have achieved elimination are needed to understand the effect of waning total rubella IgG levels in the population. In light of changing epidemiology of universally vaccinated populations, and the variability between rubella IgG assays, it may therefore be appropriate to once again review the current indicators for immunity, and reexamine the role for neutralization assays in population screening.

Effect of laboratory testing on public health and health care costs

While acute rubella infections are typically benign, with symptoms including fever, headache, lymphadenopathy, rash and pharyngitis, if rubella is acquired early in pregnancy, the fetus is at high risk to develop CRS.¹⁹ Fetal sequelae from CRS can be severe, and include limb aplasia, blindness, decreased birth weight, and deafness.^20,21 Screening women preconception, or early in pregnancy, is therefore important to identify those who are at risk for rubella infection. Routinely, prenatal screening is performed in the first trimester where total rubella IgG antibody levels are measured.³ Based on the 1992 NCCLS Rubella Subcommittee recommendations, women who have antibodies >10 IU/ml should be considered immune, while those with antibody levels ≤10 IU/ml should be considered susceptible to infection. This generates questions from the laboratory and physicians alike for women who are only slightly below the cutoff. Should a woman receive postnatal vaccination if antibody levels of 9 IU/ml are measured? And is she protected from rubella infection for her current pregnancy? Most countries with universal childhood rubella vaccination may not have actively circulating virus in the communities, therefore the chance of encountering the virus, and therefore of subsequent infection, is small. However what would happen if a woman were to travel to an area that is endemic for rubella, would an antibody level of 7 IU/ml be sufficient for protection from infection? Unfortunately, vaccination of women who are pregnant is contraindicated,²² therefore pregnant women with rubella IgG antibody levels <10 IU/ml can only be vaccinated postnatally. Postnatal vaccination will offer protection during subsequent pregnancies, however it will not protect the current pregnancy. From a public health perspective, all women who test <10 IU/ml should be vaccinated postnatally, but it is difficult to offer advice to a currently pregnant women as to her immune status when she is <10 IU/ml. Mathematical modeling indicated that the 10 IU/ml cut off in a prenatal population of 50,000 would capture all women who were susceptible to infection (700), but it would also capture 5,936 women who have detectable antibody levels, while a cut off of 5 IU/ml would capture all women susceptible to infection, and only capture 1,307 women who have detectable antibody levels.³ These results are interesting in light of the changing epidemiology in universally vaccinated populations, however more extensive analyses are needed to determine whether those women with low, but detectable antibody levels (i.e. those <10 IU/ml) would have sufficient level of neutralizing antibody to be protected from rubella infection.

The cost of an inappropriate antibody cut off to public health is significant. If a cut off is too low, the probability for CRS could increase because women who lack a protective immune response may be considered protected from infection. However, when cut offs are set higher than the epidemiology of the population suggests is protective, many women who are immune to infection are offered, and receive, postnatal vaccination inappropriately. In our health care system from 2009–2015, all women screened for rubella IgG in their first trimester who were <15 IU/ml were offered postnatal vaccination. The cost to offer this, including patient letters to explain the benefit for vaccination, appointment costs, and vaccination cost, is significant, with an approximate cost of $199,000 CAD per year. Latent class modeling of this population suggested that a cut off of 10 IU/ml, with an indeterminate range of 5–10 IU/ml, would more accurately reflect immunity to rubella in our population, without misclassification of women who lack immunity to rubella.³ Based on our testing numbers for one year, the change in the cut off from 15 to 10 IU/ml would save approximately $85,000 CAD per year in follow up of women who have immunity to rubella. It may be prudent for laboratories to examine their testing population, including the level of vaccination to determine if current rubella antibody cutoffs are appropriate for their population base. However, more studies are needed to correlate true protection in the era of universal childhood rubella vaccination, and to understand the significance of waning antibody levels in a population.

Conclusion

In light of the changing epidemiology of universally vaccinated populations, it may be time to reassess the level of antibody that indicates immunity to rubella infection. Waning antibody levels over time have been well documented in countries offering universal vaccination, and the cost for patient follow up is increasing for Public Health providers. Accurate identification of women who lack a protective immune response to rubella, and appropriate follow up of these women may be as essential as accurate identification of women who have immunity to rubella, to prevent unnecessary follow up and intervention by Public Health teams. More work is needed in populations that have achieved rubella elimination to determine what antibody value would accurately indicate immunity to infection.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.