Immunity to six vaccine pathogens in HIV positive and HIV negative children in Equatorial Guinea

Abstract

Vaccination prevents millions of childhood deaths annually, yet 23 million infants still miss essential vaccines, mostly in Sub-Saharan Africa. Low coverage is particularly concerting in countries with high paediatric HIV prevalence. In Equatorial Guinea, where 6% of children live with HIV and vaccination coverage remains limited, information on vaccine-induced immunity is scarce. We conducted a cohort study to evaluate immunity against six vaccine-preventable diseases in HIV-exposed children from Bata. Plasma samples from 69 children (55 HIV+/14 exposed uninfected) collected between 2019 and 2021 were analysed by a chemiluminescent immunoassay. Despite antiretroviral therapy, only 18.5% of HIV + children achieved viral suppression. Reported vaccination coverage for DTP and measles was lower than WHO-UNICEF estimates. IgG antibody levels was 68.1% for diphtheria, 13% for tetanus, 5.8% for pertussis, and 49.3% for measles. Although mumps and rubella vaccines are not included in the national schedule, antibody prevalence was 79.7% and 59.4%, respectively, suggesting natural exposure. Vaccination records were incomplete for half of the children. HIV-exposed children in EG show insufficient antibody levels against major vaccine-preventable pathogens. Strengthening immunization services, improving vaccine record systems, and tailoring strategies for HIV-exposed children are critical priorities.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-28643-w.

Introduction

Immunisation through vaccines is the main public health measure to reduce morbidity and mortality in children. Currently, it prevents between 3.5 and 5 million deaths worldwide each year from diseases such as diphtheria, tetanus, pertussis, influenza, and measles¹. Despite these advances, around 20 million children still do not receive essential vaccines, such as the diphtheria-tetanus-pertussis-containing vaccine (DTP) or the measles vaccine². In 2015, the Global Vaccine Action Plan set the goal of reaching at least 90% national coverage for the 3-dose schedule of DTP and extending it to all vaccines included in each country’s immunisation programs by 2020¹. However, vaccination coverage has stagnated in recent years and even declined since 2020, particularly in Southeast Asia and Sub-Saharan Africa, partly due to the COVID-19 pandemic, putting children at higher risk of vaccine-preventable diseases.

Equatorial Guinea (EG), a small country located in West Central Africa, had an under-five mortality rate of 77 per 1000 live births in 2021^3,4, mainly due to malaria, HIV, and acute respiratory or diarrheal infections. According to WHO data, national coverage for DPT3 and measles-containing-vaccine (MCV) in 2022 was only 53%⁵, far below the global average of 81%¹, with no data on rubella and mumps vaccine coverage since their vaccines are not in the current EG vaccination schedule. These percentages have remained unchanged since 2016⁶. The introduction of a second MCV dose in 2021, has so far resulted in very low uptake (13% in 2022). The lack of proper immunisation in the paediatric population can lead to periodic outbreaks of vaccine-preventable diseases. Incidence data in EG are scarce: annual figures are unavailable for diphtheria, tetanus, pertussis, and mumps, while only 49 measles and 47 rubella cases were reported in 2022⁶. Weaknesses in the national surveillance and reporting system have been acknowledged, underscoring the need for additional approaches such as household surveys and serological studies. To date, no study has assessed the seroprevalence of major vaccine-preventable diseases in Equatoguinean children.

This issue is particularly relevant given EG’s high HIV burden. With a national prevalence of 6.6% - the highest in its WHO region - an estimated 72.000 people are living with HIV, of whom 6% are children⁷. Children exposed to HIV, whether infected or not, are a uniquely vulnerable group. HIV infection is known to reduce vaccine-induced immunity due to immune suppression, while HIV-exposed but uninfected children also show higher risk of morbidity and impaired vaccine response⁸.Optimising the current vaccination schedule (Suppl. Figure 1) and regularly monitoring coverage is therefore crucial in this country, particularly for HIV-infected children. IgG antibody levels following immunisation in HIV-positive children vary by vaccine type, immune recovery, and viral suppression⁹. Moreover, revaccination after antiretroviral therapy (ART) may help to improve immune protection¹⁰. These factors highlight the importance of assessing vaccine-derived immunity, specifically in HIV-exposed paediatric populations in EG.

The aim of this study was therefore to determine the proportion of HIV-exposed Equatoguinean children with specific IgG antibodies against key vaccine-preventable diseases. In parallel, we reviewed available data on vaccination coverage for other WHO-recommended vaccines in EG, such as yellow fever, oral polio (OPV) and tuberculosis (BCG), to provide broader context for interpreting our findings.

Methods

Study design and population

Plasma samples were collected between 2019 and 2021 from 69 HIV-exposed children (55 HIV-positive and 14 HIV-exposed but uninfected), all born to HIV-positive mothers and with a median age of 8.25 years, who were under clinical follow-up at the General Hospital of Bata (EG)(Suppl. Table 1). These samples were part of a larger study investigating antiretroviral resistance in paediatric and adult HIV-positive populations in Equatorial Guinea¹¹. The children were selected by the attending paediatricians during routine consultations, and all clinical data associated with each sample were recorded by medical staff based on the information available at the time of the visit. 80% of the children were HIV+, while 20% were HIV-negative. According to the information contained in the medical reports, 36 (52.9%) of the 69 children had complete vaccination information available regarding the six analysed diseases, while 33 (47.9%) had no vaccination information available. Among these, 86.1% and 72.7% respectively were HIV+. Patients’ names were codified at sampling to maintain confidentiality. We considered correctly vaccinated children if they had received all doses of each of the vaccines recommended in Equatorial Guinea vaccination schedule on time, accordingly to medical reports. The specific vaccines, number of doses, and recommended ages for administration are detailed in the Suppl. Figure 1.

Sample collection and processing

EDTA-anticoagulated blood samples were obtained through venepuncture and centrifuged at 3,000 rpm for 10 min to separate plasma from blood cells. These samples were initially collected for routine analysis. After completion, we used the remaining samples for this and other research projects¹¹. The plasma was stored in cryotubes at −20 °C until transport to Madrid.

HIV diagnostic confirmation

The first HIV diagnosis was performed in Bata with two rapid serological tests in children older than 18 months of age¹². No diagnostic tests were performed in children under 18 months of age due to the unavailability of molecular tests in the country. The serological status of the patients using the Geenius™ HIV-1/2 (Bio-Rad) serological assay and the molecular point-of-care Xpert Qual HIV (Cepheid) in patients under 18 months was carried out at the HIV-1 Molecular Epidemiology Laboratory at Ramón y Cajal Hospital (Madrid, Spain). Out of 55 HIV-exposed children classified as HIV + in Bata, 54 were HIV-confirmed in Madrid, with only one case identified as a false positive. Only one HIV-exposed child out of 14 was found to be HIV+. Both HIV-exposed children were reclassified into the correct group after definitive diagnosis in Madrid; the false positive in the HIV-negative group and the confirmed positive in the HIV-positive group.

Antibody detection and interpretation

IgG against diphtheria, tetanus, and pertussis (present in the DTP vaccine), as well as measles, mumps, and rubella acquired through natural infection or vaccination were detected using the VirClia^® automated system (Vircell), an indirect chemiluminescent immunoassay (CLIA), which uses only 5 µL of plasma volume. We used as gold standard the cut-off values provided by VirClia^® for the six pathogens in plasma/serum (Suppl. Table 2), which provide 96–100% sensitivity and 100% specificity. We considered that a child had specific IgG levels for a pathogen under study, or concentration of antibody titers with sufficient affinity required to neutralize the pathogen, when the anti-pathogen IgG levels in his plasma were included in the range of positivity proposed by Vircell, and specified in the technical file of each specific VIRCLIA^® IgG assay. This technique does not allow differentiation between neutralising and non-neutralising antibodies (or binding antibodies). Therefore, we can only assess levels of IgG antibodies (through natural infection or vaccination) and not levels of protection due to vaccination. Despite not being included in the current EG vaccination schedule, we also studied IgG antibody levels against mumps, and rubella since they are two of the three pathogens included in the highly effective MMR vaccine.

Statistical analysis

Descriptive statistics were calculated using Microsoft Excel and GradPad-Prism statistical software. Statistical differences between group 1 (patients vaccinated with at least one vaccine included in the country’s vaccination schedule according to their clinical files) and group 2 (unvaccinated patients or those lacking vaccination information in their medical records) were assessed using the Chi-square or Fisher’s exact test, when the sample size of any category was less than five. All variables analysed were categorical, including serological status for each pathogen (positive/negative), HIV status (positive/negative) and vaccination status (vaccinated/unvaccinated or unknown). No analyses were conducted using continuous variables, such as age or antibody titre, as the main objective of the study was to describe the presence or absence of specific antibodies.

Ethical approval statement

The authors confirm that all experiments have been conducted in accordance with relevant guidelines and regulations. The study was conducted accordance with the Declaration of Helsinki and approved by the local Ethics Committee for Clinical Investigation from Hospital Universitario Ramón y Cajal (Madrid, Spain) (ACTA 369, 24 June 2019) and the Ministry of Health and Social Welfare of Equatorial Guinea. Signed informed consent was obtained for all parents or legal guardians of children included in the study cohort.

Results

Study population

The median age of the 69 patients in the study population was 8.25 years, and all were born to HIV-positive women. The characteristics of the study population are detailed in Table 1.

Table 1.

Characteristics of the study population according to vaccine-related information recorded in their clinical files.

		Group 1	Group 2
Vaccine-related information in clinical files		Vaccinated*	Not vaccinated	No information	Whole study population
n (%)		34 (49.3%)	2 (2.9%)	33 (47.8%)	69 (100%)
Median age at sampling in years [IQR]		7.5 [0.11–17.11]	8 [6–10]	9 [0.08–17.08]	8.25 [0.08–17]
HIV-positive		29 (85%)	2 (100%)	24 (72%)	55 (79.7%)
Viral load among HIV-positive (plasma)
Undetectable (< 40cp/mL)		1 (3%)	0	2 (8.3%)	3 (5.4%)
40cp/mL < VL < 1000cp/mL		6 (21%)	0	1 (4.2%)	7 (12.7%)
VL > 1000cp/mL		22 (76%)	2 (100%)	21 (87.5%)	45 (81.8%)
Reported vaccination coverage data in clinical files
DTP*+HB + Hi		30 (88.2%)	0	unknown	30 (43.5%)
Measles		26 (76.5%)	0	unknown	26 (37.7%)
BCG		34 (100%)	0	unknown	34 (49.3%)
OPV		25 (73.5%)	0	unknown	25 (36.2%)
Yellow Fever		9 (26.5%)	0	unknown	9 (13%)
Pathogen immunity by VirClia in plasma
Diphtheria IgG (%)		24 (70.6%)	1 (50%)	22 (66.7%)	46 (68.1%)
Tetanus IgG (%)		5 (14.7%)	0	4 (12.1%)	9 (13%)
Pertussis IgG (%)		4 (11.8%)	0	0	4 (5.8%)
Measles (%)		18 (52.9%)	1 (50%)	15 (45.4%)	34 (49.3%)
Mumps (%)		27 (79.4%)	2 (100%)	26 (78.8%)	55 (79.7%)
Rubella (%)		21 (61.8%)	1 (50%)	19 (57.6%)	41 (59.4%)

*Vaccinated with at least one vaccine included in Equatorial Guinea vaccination schedule. N, number of children; %, percentage; VL, Viral load; cp/mL, HIV-1 RNA copies per plasma millilitre. DTP*, unknown DPT doses; HB, Hepatitis B; Hi, Haemophilus influenzae type b; BCG, Bacillus Calmette-Guérin vaccine; OPV, oral poliovirus vaccine. Vaccination coverage data according to clinical files in Bata Hospital. Age data was calculated among subjects 61/69 (89%) with available data.

Vaccination coverage based on clinical records

Out of the 69 children under study, only 34 (49.3%) had been vaccinated against at least one of the six analysed pathogens according to the clinical files, while two children (2.9%) had not received any of the six vaccines. However, in 33 children (47.8%), vaccine-related information was absent in the clinical files. In those reported as DTP vaccinated, the number of DTP doses was not indicated.

The vaccination coverage observed in the paediatric population under study, regardless of age or HIV infection, was lower than the figures reported by WHO-UNICEF for the corresponding years⁵. This trend was consistent across all pathogens with documented vaccinations in the country (Fig. 1). Vaccination coverage against DTP was 43.5%, and for measles, it was 37.7%, in contrast to the 53% reported by WHO for both vaccines.

Fig. 1.

Reported vaccine coverage in the study population vs. WHO-UNICEF for the years 2019-2021. BCG, Bacillus Calmette-Guérin vaccine; DTP+HB+Hi, diphtheria/tetanus/pertussis/hepatitis B/Haemophilus Influenzae type b combined vaccine; OPV, oral poliovirus vaccine.

Specific IgG antibody detection against the six vaccine-preventable diseases studied

When examining the percentage of children with IgG in plasma against each of the six analysed pathogens in the overall study population, regardless of vaccine-related information in their clinical files, we observed significant variability among the pathogens. Specifically, 79.7% of them exhibited IgG levels against mumps, 59.4% for rubella, 68.1% for diphtheria, 49.3% for measles, 13% for tetanus, and 5.8% for pertussis (Fig. 2). A notable percentage of indeterminate results for tetanus (23.2%) remained unresolved, while for the remaining pathogens, it was less than 6%.

Fig. 2.

Presence of specific IgG against the six vaccine-preventable diseases in the study population (n=69). The plasma cut off provided by Vircell for each pathogen is shown in Suppl. Table 2.

Specific IgG antibody levels by vaccination status

The rate of children with IgG antibody levels in plasma against mumps, rubella, measles, tetanus, and diphtheria was similar among Groups 1 (vaccinated children, n = 34) and 2 (unvaccinated or unknown status, n = 35), with no statistically significant differences observed (p > 0.05 for all comparisons) (Table 1). Specifically, IgG seropositivity for measles was found in 57.7% of children in Group 1 vs. 44.2% in Group 2 (p = 0.276), and for diphtheria 67% vs. 69.2% (p = 0.821). A statistically significant difference was observed in IgG levels against pertussis: 13.3% of children in Group 1 had detectable IgG, compared to 0% in Group 2 (p = 0.0317, Fisher’s exact test), being the only pathogen for which seropositivity was exclusively to the vaccinated group (Fig. 3). Regarding tetanus, IgG positivity was low in both groups (13.3% in Group 1 vs. 12.8% in Group 2), with no significant difference (p = 0.95), and a high percentage of indeterminate results.

Fig. 3.

Specific IgG in two groups of children according to their reported vaccination status. The plasma cut off provided by Vircell for each pathogen is shown in Suppl. Table 2. INDET, undetermined results according to the manufacturer’s instructions. Description of groups in Table 1. **, p<0.01; *, p<0.05.

Discussion

Our findings reveal concerningly low levels of specific IgG antibodies against several vaccine-preventable diseases in HIV-exposed children in Equatorial Guinea, indicating significant susceptibility to infection, despite existing vaccination programmes. In particular, antibody levels against measles, tetanus and pertussis were markedly below the expected thresholds for herd immunity, while antibodies to mumps and rubella appeared to derive mainly from natural exposure rather than vaccination. These results underscore major gaps in vaccine coverage and highlight the urgent need to strengthen immunisation strategies in EG.

Protection against disease is not solely determined by the development of neutralising antibodies against a specific pathogen (humoral immunity) but also relies on the innate, cellular or cytokine response. In our study, we have focused on the quantification of IgG antibody levels to analyse the exposure and probable immunity of children in our EG study population to six of the most important vaccine-preventable pathogens worldwide: diphtheria, tetanus, pertussis, measles, rubella, and mumps.

Half (50%) of Equatoguinean children in our study population had measles-specific IgG antibodies, a level far below the herd immunity threshold of 92–94%¹³, and a vaccination coverage of 37.7%. This finding is consistent with the low national measles-containing vaccine (MCV) coverage of 53% reported by WHO⁶ and is further exacerbated by the absence of a second MCV dose in our study population, which has only been introduced in EG since 2021. Together, these results highlight the high risk of measles outbreaks and the urgent need to improve measles vaccine uptake and completion. For mumps and rubella, specific antibody prevalence was unexpectedly high (around 80% and 60% respectively), despite the absence of these vaccines from the EG immunization schedule¹⁴. Even so, they still do not reach the threshold for herd immunity (83%−85% for rubella and 75–86% against mumps)¹³. This strongly suggests that natural infection is the main source of immunity, which carries an increased risk of outbreak among the unprotected paediatric and adolescent population. The situation is particularly concerning for rubella, as outbreaks can have severe consequences for women of childbearing age and newborns. It is also important to consider that HIV-exposed children may mount weaker or less durable vaccine-induced responses, and their maternal transfer of antibodies may be compromised, further exacerbating their vulnerability to measles and rubella epidemics. These findings underscore the vulnerability of the HIV-exposed pediatric population to epidemics of measles, mumps, and rubella, and highlight critical gaps in the current immunisation strategy in EG.

Antibody prevalence differed markedly across the DTP components: nearly 70% of children had specific IgG levels against diphtheria, compared with only 13% for tetanus and 5.8% for pertussis. These proportions are lower than the national DTP3 coverage reported by the WHO for EG or by clinical files in the study population, which would indicate that 4–5 out of 10 children should have received the DTP vaccine, highlighting a gap between reported vaccination and effective immunity. The presence of antibody levels exceeding coverage estimates for diphtheria would indicate that some children might have acquired immunity through natural exposure to the pathogen, a process that carries important health risks¹⁵. The low prevalence of pertussis antibodies would be consistent with waning effect after early vaccination, as previously reported¹⁶. Tetanus requires particular attention, since herd immunity does not apply: C. tetani is environmentally acquired, making individual protection essential. The very low proportion of children with specific tetanus antibodies (10%) was especially concerning, as neonatal tetanus in resource-limited settings can be almost universally fatal without vaccination¹⁷. The particularly low persistence of tetanus and pertussis antibodies in our HIV-exposed cohort is consistent with prior evidence that these children experience reduced vaccine immunogenicity and more rapid waning of protective responses, underscoring the need for tailored booster strategies. These findings underscore the urgent need to strengthen DTP immunisation strategies and booster implementation among HIV-exposed children in EG.

HIV exposure is a key factor that may explain part of the suboptimal antibody levels observed in our study population. The increasing success of prevention of mother-to-child HIV transmission programmes in África has led to a large and growing population of HIV-exposed but uninfected children. These children face higher risks of morbidity, mortality, and impaired growth compared to their HIV-unexposed peers¹⁸, likely due to increased inflammation and monocyte activation¹⁹, as well as reduced T-cell functionality during pregnancy²⁰. It is known that children living with HIV tend to develop lower vaccine-induced immunity due to their immunosuppression than HIV-exposed or healthy children²¹. Furthermore, the reduced immunogenicity of vaccines observed in HIV-positive pregnant women may compromise the passive transfer of protective antibodies to their infants²². Our findings of low antibody levels across several vaccine-preventable diseases fit into this broader evidence, highlighting the particular vulnerability of HIV-exposed children in EG.

Taken together, our antibody data in EG demonstrated a large gap between the observed immunity levels and the herd immunity thresholds required to prevent outbreaks of measles, rubella, mumps, diphtheria, pertussis, and tetanus^23–25. This situation underscores the urgent need for enhanced immunisation strategies in EG. The lack of accurate vaccination records complicates the interpretation of both coverage and immunogenicity data, as underreporting and under-administration cannot be easily distinguished. These weaknesses in registry systems are critical to address, since they directly impact the planning and monitoring of vaccination campaigns. Importantly, EG is currently not on track to meet Sustainable Development Goal 3 of reducing child mortality below 25 per 1000 live births by 2030, with projections suggesting this target may not be achieved until after 2050³. Factors such as development aid, maternal education and good governance have been linked to reduced vaccination inequalities, while investing in immunisation programmes, especially for HIV-exposed and other vulnerable children, is an urgent public health priority.

Several limitations should be considered when interpreting our findings. First, the use of VirClia, a non-neutralising assay, means we cannot confirm the protective nature of the antibodies measured, which prevents us from distinguishing between antibodies derived from vaccination and those resulting from natural exposure. Thus, we cannot distinguish neutralizing from non-neutralizing antibodies, as viral neutralization assays (for measles, mumps, or rubella) or growth inhibition assays (in the case of bacteria such as diphtheria, tetanus, and Bordetella pertussis) were not conducted. Thus, IgG positivity should not be interpreted as definitive protection. This limitation is particularly relevant for pathogens with available vaccines in the country, such as diphtheria, tetanus, pertussis, and measles. Second, we only measured IgG levels and did not assess cell-mediated immunity, which is also essential for comprehensive protection. Third, our study population consisted exclusively of HIV-exposed children (n = 69), which limits generalisability to the broader paediatric population. The inclusion of infants younger than one year further complicated interpretation, as maternal antibodies can persist up to 18 months and may have contributed to observed IgG levels²⁶. Moreover, we were unable to directly compare HIV-positive and HIV-negative children due to significant age differences between the groups. The absence of a well-balanced HIV-negative control group limits the interpretation of immunological differences between immunocompetent and immunocompromised children. Fourth, vaccination history was incomplete in clinical files, with about half of the children lacking sufficient information and making it uncertain whether reported DTP vaccinations reflected full 3-dose completion. This complicated the interpretation of coverage and raised the question of whether the discrepancies observed with WHO-UNICEF estimates could reflect under-reporting or under-administration of vaccines in EG. It is important to note that Equatorial Guinea currently lacks a centralised electronic medical record system, which limits the access to complete medical histories.

These factors collectively highlight the need for future studies with larger, more representative cohorts, detailed vaccination records, and the inclusion of HIV-negative exposed and unexposed control groups, to enable more comprehensive comparisons. Such studies should also consider other vaccine-preventable pathogens included in the vaccination schedule of EG, such as hepatitis B and Haemophilus influenzae type b. Even so, our findings underscore the importance of ensuring adequate immunisation among HIV-exposed and HIV-positive children to reduce susceptibility to vaccine preventable diseases and to improve overall health outcomes.

In conclusion, our study shows that HIV-exposed children in EG have insufficient specific antibody levels against multiple vaccine-preventable diseases, with levels well below herd immunity theresholds. This immunological gap places children at continued risk of outbreaks and severe disease, especially in the context of fragile healthcare systems. Strengthening paediatric immunisation strategies, with particular attention to HIV-exposed populations, is therefore an urgent public health priority. Equally important are the establishment of reliable vaccination records and the implementation of periodic sero-surveillance, which would allow accurate monitoring of immunisation coverage and inform targeted interventions. Achieving and sustaining high vaccination coverage is essential to protect the health of children in EG and to advance towards global targets for child survival and health equity.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, study design and proposal writing, Á.H.; Methodology, Á.H., A.R.-G. and J.V.-C.; Patient’s selection in Equatorial Guinea, M.E.; Sample collection in Equatorial Guinea, T.M.-O. and J.N.; Coordinator in Equatorial Guinea, J.N. and P.N.; Sample shipping coordinator, P.N. and A.B.; Experimental procedures in Spain, A.R.-G. and J.V.-C.; Results supervision, Á.H.; Results discussion, A.R.-G., J.V.-C. and Á.H.; Writing-original draft preparation and final version editing, A.R.-G. and Á.H.; Writing-review, A.R.-G., A.D and Á.H.; Project coordinator in Spain, Á.H. and A.B.; Project administration, resources and funding acquisition, Á.H. and A.B. All authors have read and agreed to the published version of the manuscript.

Data availability

The data provided in the manuscript and the supplementary files are accurate and complete and have not been manipulated in any way. Information on data availability for review by other investigators will be provided upon request to Dr. África Holguín (africa.hoguin@salud.madrid.org).

Declarations

Competing interests

The authors declare no competing interests.