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\begin{document}

\title{Study of pneumococcal vaccine response in children with underlying
medical conditions}



\author[1]{Marta Garrido Jareño}%
\author[1]{José Miguel Sahuquillo Arce}%
\author[1]{Hector Rodríguez Vega}%
\author[1]{Carmen Lloret Sos}%
\author[1]{Ana Gil Brusola}%
\author[1]{José Luis  López Hontangas}%
\author[1]{Carmen Carreras Vázquez}%
\author[2]{Leonor Puchades Carrasco}%
\author[3]{Antonio Pineda Lucena}%
\author[2]{Javier Peman García}%
\affil[1]{La Fe University and Polytechnic Hospital}%
\affil[2]{Health Research Institute La Fe}%
\affil[3]{Centre for Applied Medical Research}%


\vspace{-1em}



  
  \date{June 06, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Pneumococcal infection causes serious illness in children,
especially those with underlying conditions. Although pneumococcal
vaccination in the pediatric population is recommended, the immune
response to pneumococcal vaccines in this group is usually deficient.
The aim of this study was to study the response to pneumococcal vaccines
in children with underlying conditions. Methods: A one-year
retrospective study on children with underlying conditions attended at
the Pediatric Immunology Unit of La Fe University Hospital (Valencia,
Spain). Patient demographics, immunosuppressive diseases, pneumococcal
vaccination coverage, serum levels of C3, C4, IgA, IgM, IgG, IgG1, IgG2,
IgG3, IgG4, blood levels of leukocytes, neutrophils and CD4 and CD8 T
lymphocyte were analyzed. Results: A total of 85 children were included
in the study. Only 29.41\% of them had the complete vaccination
schedule, most under 5 years of age (76.00\%). Protective levels of
pneumococcal antibodies were found only in 48\% of the correctly
vaccinated patients, whose serum levels of IgG4 and CD4/CD8 ratio were
higher than in children with deficient response. Low vaccine response
was mostly found among leukemic patients, who exhibited the lowest level
of CD4 T lymphocyte. Patients' age, immunosuppressive therapy, and serum
levels of C3, C4, IgG3 and lymphocytes were associated with pneumococcal
vaccine response. Conclusion: Most of the patients lacked some dose of
pneumococcal vaccine and had an inadequate response. Mathematical models
can be useful in clinical practice to detect non responders. Emphasis
should be placed on completing a vaccination schedule and, if necessary,
revaccination with a booster dose of pneumococcal vaccines.%
\end{abstract}%



\sloppy


\textbf{Study of pneumococcal vaccine response in children with
underlying medical conditions}

Marta Garrido-Jare\selectlanguage{ngerman}ño, MSc\textsuperscript{1,2,3*}, José Miguel
Sahuquillo-Arce, MD\textsuperscript{2,4}, Héctor Rodríguez-Vega,
MD\textsuperscript{5}, Carmen Lloret-Sos, MD\textsuperscript{2}, Ana
Gil-Brusola, MD, PhD\textsuperscript{2,6}, José Luis López-Hontangas
MSc\textsuperscript{2,6}, Carmen Carreras-Vázquez,
MD\textsuperscript{5}, Leonor Puchades-Carrasco,
PhD\textsuperscript{1,}Antonio Pineda-Lucena, PhD\textsuperscript{7},
Javier Pemán-García, MD, PhD\textsuperscript{2,6}

\textsuperscript{1}Drug Discovery Unit, Health Research Institute
Hospital La Fe (Valencia, Spain)

\textsuperscript{2}Microbiology Department, University and Polytechnic
Hospital La Fe (Valencia, Spain)

\textsuperscript{3}Valencian Institute of Pathology, Catholic University
of Valencia (Valencia, Spain)

\textsuperscript{4}Respiratory Infection Research Group, Health Research
Institute Hospital La Fe (Valencia, Spain)

\textsuperscript{5}Paediatric Department, University and Polytechnic
Hospital La Fe (Valencia, Spain)

\textsuperscript{6}Severe Infection Research Group, Health Research
Institute Hospital La Fe (Valencia, Spain)

\textsuperscript{7}Molecular Therapeutics Program, Center for Applied
Medical Research, University of Navarra (Pamplona, Spain)

\textbf{Correspondence*}

Marta Garrido Jareño

Microbiology Department, University and Polytechnic Hospital La Fe
(Valencia, Spain)

Avenida Fernando Abril Martorell, 106 Torre A 3ª planta

46026 Valencia

\textbf{Telephone number} : 34 692933193

\textbf{E-mail} : marta.garrido@ucv.es

\textbf{Key words:} Pneumococcal vaccine; Children with underlying
conditions; Vaccination status; Immunoglobulin levels; Prediction of
vaccine response.

\textbf{Abstract}

\textbf{Background:} Pneumococcal infection causes serious illness in
children, especially those with underlying conditions. Although
pneumococcal vaccination in the pediatric population is recommended, the
immune response to pneumococcal vaccines in this group is usually
deficient. The aim of this study was to study the response to
pneumococcal vaccines in children with underlying conditions.

\textbf{Methods:} A one-year retrospective study on children with
underlying conditions attended at the Pediatric Immunology Unit of La Fe
University Hospital (Valencia, Spain). Patient demographics,
immunosuppressive diseases, pneumococcal vaccination coverage, serum
levels of C3, C4, IgA, IgM, IgG, IgG\textsubscript{1},
IgG\textsubscript{2}, IgG\textsubscript{3}, IgG\textsubscript{4}, blood
levels of leukocytes, neutrophils and CD4 and CD8 T lymphocyte were
analyzed.

\textbf{Results:} A total of 85 children were included in the study.
Only 29.41\% of them had the complete vaccination schedule, most under 5
years of age (76.00\%). Protective levels of pneumococcal antibodies
were found only in 48\% of the correctly vaccinated patients, whose
serum levels of IgG4 and CD4/CD8 ratio were higher than in children with
deficient response. Low vaccine response was mostly found among leukemic
patients, who exhibited the lowest level of CD4 T lymphocyte. Patients'
age, immunosuppressive therapy, and serum levels of C3, C4, IgG3 and
lymphocytes were associated with pneumococcal vaccine response.

\textbf{Conclusion:} Most of the patients lacked some dose of
pneumococcal vaccine and had an inadequate response. Mathematical models
can be useful in clinical practice to detect non responders. Emphasis
should be placed on completing a vaccination schedule and, if necessary,
revaccination with a booster dose of pneumococcal vaccines.

\textbf{INTRODUCTION}

Pneumococcal infection, especially invasive pneumococcal disease (IPD),
includes potentially fatal conditions such as meningitis, bacteremia or
pneumonia, and it accounts for about 11\% of all deaths worldwide in
children under 5 years of age\textsuperscript{1}. According to WHO
reports\textsuperscript{2}, \emph{Streptococcus pneumoniae} caused about
14.5 million cases of serious illness and 735,000 deaths in non-HIV
children under 5 in the year 2000. Regardless of age, the most
vulnerable groups comprise mainly patients who are immunocompromised due
to splenectomy, drepanocytosis, bone marrow transplant, HIV infection,
chronic inflammatory diseases or are under immunosuppressive treatment
(IT)\textsuperscript{3,4}.

Pneumococcal vaccines have directly decreased the rates of IPD on
vaccinated children and indirectly --through herd immunity-- on
unvaccinated children\textsuperscript{5}. However, the immune response
to pneumococcal vaccines in immunocompromised children (IC) is
reportedly lower than in healthy children, especially when conjugated
vaccines are employed\textsuperscript{6}.

Consequently, the Advisory Committee on Vaccines (ACV) of the Spanish
Association of Pediatrics recommends special vaccination schedules for
IC based on the routinely use of pneumococcal conjugated vaccines in
combination with booster doses of polysaccharide vaccines, but also for
immunocompetent children with underlying chronic medical
conditions\textsuperscript{7}. The main difference being the greater
number of pneumococcal vaccine shots received by this particular group
of patients (Table S1\textsuperscript{7}).

Despite the extensive use of pneumococcal vaccines worldwide, measuring
pneumococcal antibody responses is still problematic, especially the
assessment and detection of serotype-specific
antibodies\textsuperscript{8}. In addition, immune responses present
considerable inter-individual variation, particularly in IC, and some
studies relate pneumococcal vaccine failure to
co-morbidities\textsuperscript{9,10}.

Cytokines, major immunoglobulin classes (IgG, IgA, IgM), complement
system and white blood cells play central roles in a proper protective
immune response, including that to pneumococcal
vaccines\textsuperscript{11}. Thus, serum levels of these components
could be used to identify low or non-responder patients who could
benefit from an alternative vaccination schedule.

Systems biology approaches and mathematical modelling are increasingly
being used to improve the understanding and the prediction of biological
processes\textsuperscript{12,13}. Several models to estimate the impact
of the immunogenicity and safety of pneumococcal vaccination in IC
patients have been published over the last few years, particularly on
PCV13\textsuperscript{14-16}; but, the relationship between the serum
proteins involved in the immune response and the pneumococcal vaccine
should be studied in more detail to assist clinical management,
particularly in IC and in children with underlying conditions. Under
this rationale, we have developed a mathematical model to predict
pneumococcal vaccine responses in children with previous immunological
alterations to guide clinical decision-making based on the baseline
immunological status of each patient and reach a personalized medicine.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1 Study design}

An observational retrospective study was performed in La Fe University
Hospital from November 2018 to November 2019. Inclusion criteria were
patients under 16 years of age attended at the Pediatric Immunology Unit
who had had a pneumococcal serology performed during the study period.

Children were classified in 8 groups according to their main clinical
diagnosis: a) Chronic pulmonary disease (CPD): bronchiectasis,
bronchitis and asthma; b) Recurrent non-pulmonary infections (RI):
diarrhea, meningitis and blood stream infections; c) Disorders of blood
components (DBC): sickle cell anaemia, thrombocytopenic purpura and
neutropenia; d) Leukemia and hematopoietic cell transplantation (LHT);
e) DiGeorge syndrome (DS); f) Primary immunodeficiency (PI):
hypogammaglobulinemia, chronic granulomatous disease and
ataxia-telangiectasia; g) Autoimmune diseases (AD): Crohn's disease and
severe atopic dermatitis; and h) Miscellanea (MIS); prolonged fever,
facial cellulitis, Osler-Weber-Rendu Disease, lymphadenopathies and
Turner-Syndrome.

The analysis of serum samples was performed at the Microbiology
Department and the Clinical Analysis Department. The clinical
information on the pneumococcal vaccination coverage of the patients was
obtained from the Nominal Vaccine Registry (NVR) as registered by the
Conselleria de Sanitat of the Generalitat Valenciana. Patient
recruitment and sampling procedures were performed in accordance with
the Declaration of Helsinki and applicable local regulatory requirements
and laws after approval from the Ethics Committee of the Health Research
Institute Hospital La Fe.

The database included patient demographics, clinical data associated
with the diagnosis (i.e., immunosuppressed disease, IT, previous IPD,
pneumococcal vaccination coverage, etc.), and analytical data (serum
levels of C3, C4, IgA, IgM, IgG, IgG\textsubscript{1},
IgG\textsubscript{2}, IgG\textsubscript{3}, IgG\textsubscript{4}; and
blood levels of leukocytes, neutrophils and CD4 and CD8 T lymphocytes).

\textbf{2.2 ELISA assays}

The VaccZymeTM Anti-PCP IgG Enzyme Immunoassay Kit (The Binding Site
Group, Birmingham, UK) was used according to the manufacturer's
instructions. Patients were classified into two groups according to
their Anti-PCP IgG concentration as measured by ELISA: pneumococcal
vaccine responders (IgG \textgreater{}50 mg/L) and non-responders (IgG
\selectlanguage{english}[?]50 mg/L)\textsuperscript{17,18}.

\textbf{2.3 Statistical Analysis}

The ANOVA Test was used to compare the means of anti-PCP IgG, C3, C4,
IgA, IgM, IgG, IgG\textsubscript{1}, IgG\textsubscript{2},
IgG\textsubscript{3}, IgG\textsubscript{4}, leukocytes, neutrophils, CD4
and CD8 T lymphocytes and CD4/CD8 ratios of the 8 groups. The Student's
t-test was used to compare the means according to the vaccine response
profile in patients with correct immunization schedule. Fisher's exact
test was used to compare categorical variables.

Multiple linear regression backward elimination was performed to predict
pneumococcal vaccine response. The variables used in the analysis were
age, sex, C3, C4, IT, IgA, IgM, IgE, IgG, IgG1, IgG2, IgG3, IgG4, total
leukocytes count, total lymphocytes count, monocytes, eosinophils,
basophils, lymphocytes TCD8, lymphocytes TCD4 and CD4/CD8 ratio.\emph{P}
-values \textless{}0.05 were considered statistically significant.

\textbf{3. RESULTS}

\textbf{3.1 Results according to patient's age}

A total of 85 patients were included during the study period. The mean
age was 7.17 +- 4.76 years. More than half of the patients were boys
(67.06\%). Vaccination profiles, IPD history and anti-PCP IgG levels
according to patients' age, sex and principal diagnosis are summarized
in Table 1.

Conjugated and polysaccharide vaccines were used alternately depending
on the year of inoculation. Combinations of conjugated vaccines were
common, especially with PCV13 and PCV7 in older patients. Nevertheless,
PCV13 was the vaccine most frequently used (76.50\%), followed by PCV7
(28.24\%), PPSV23 (11.76\%), and PCV10 (2.35\%).

Six patients had been diagnosed with IPD within the previous two years,
most in the group of 10-16 years, yet one of them had a complete
immunization schedule. Patients with previous IPD did not achieve higher
anti-PCP IgG levels than patients without IPD (70.92 +- 32.54 vs 73.70 +-
77.20; T-test= 0.254; \emph{p} = 0.399).

An inverse correlation between patient's age and anti-PCP IgG titer was
observed. Older patients presented lower level of pneumococcal
antibodies, although no statistically significant differences were found
(ANOVA test = 0.951; \emph{p} = 0.417).

The immunization schedule was correct only in 25 patients (29.41\%),
nineteen of whom were under 5 years of age (76.00\%). Deficient
production of pneumococcal antibodies in patients with correct
immunization schedule was detected in 12 of these patients. Children
under 2 years presented the highest vaccination coverage rate (94.44\%);
however, almost half of them (41.17\%) were non-responders. Children
older than 2 did not reach a vaccine coverage greater than 15\%.

Most of the patients inadequately vaccinated were one dose short of
PSSV23 to properly complete the pneumococcal vaccination schedule
(85.00\%). Additionally, patients with a complete immunization schedule
did not present greater pneumococcal vaccine response rates than
children without complete immunization schedule (48.00\% vs 48.33\%,
Chi\textsuperscript{2} = 0.04; \emph{p} = 0.841).

Figure 1 represents the mean levels of serum components and CD4/CD8
ratio of the twenty-five patients with complete pneumococcal vaccination
according to vaccination response. In children with pneumococcal vaccine
response, the level of serum components was higher in all cases except
for IgG1 were it was lower, both groups being within the normal IgG1
range. Statistically significant differences were found only for IgG4 (T
Test= -2.31, \emph{p} = 0.049) and CD4/CD8 ratios (T Test=
-2.28,\emph{p} = 0.033).

\textbf{3.2 Results according to the main clinical diagnosis}

PI and RI were the most common disorders and patients were not
adequately vaccinated, regardless of the main diagnostic group (70.58\%)
However, the patients with correct vaccine response belonged mainly to
the RI group (38.46\%) followed by the PI group (23.07\%).

Patients from the RI group presented the highest level of anti-PCP IgG
(112.07 +- 109.07), whereas the lowest was found in the LHT group (25.36
+- 17.84). No statistically significant differences were found when the
means of anti-PCP IgG level of the 8 groups were compared (ANOVA test =
1.206; \emph{p} = 0.092).

Within properly vaccinated children, the LHT group presented the lower
rate of antibody response, and none of the fully vaccinated patients
achieved protective levels of anti-PCP IgG.

Of the total of patients, seventeen (20\%) were receiving IT.
Corticosteroids were the immunosuppressive drugs more frequently used
(70.6\%), followed by methotrexate (23.53\%). TNF-alpha inhibitors were
the least used (5.88\%), as summarized in Table 2.

Significant differences were found in CD4 T lymphocyte and C4 levels
among the 8 groups (ANOVA test = 3.349; \emph{p} =0.004 and ANOVA test=
12.997; \emph{p} =0.001, respectively; Figure 2A-B). RI patients had the
highest level of CD4 T lymphocyte counts while the LHT group had the
lowest. In the case of C4, the LHT group showed the highest level, while
the DBC group presented the lowest. The level of the rest of the serum
components and cell counts in each group is summarized in Tables S2 and
S3.

\textbf{3.3 Predicting pneumococcal vaccine response}

A multivariate regression model with a 67\% accuracy to predict
vaccination response was created based on patients age, IT, and blood
levels of C3, C4, IgG3 and total lymphocyte count (R=0.818; R
Square=0.670; Adjusted R Square=0.553; Std. Error of the estimate=
0.34), as is represented in Table 3. Higher age, IT and a high C3 level
contributed negatively to having an adequate pneumococcal vaccine
response while high C4, IgG3 and lymphocyte serum levels contributed
positively.

\textbf{4. DISCUSSION}

The main findings of our study are (a) pneumococcal vaccination coverage
rates in our cohort were low, (b) the LHT group presented an inadequate
response to vaccination, (c) low-pneumococcal antibody response in
patients with correct immunization schedule appeared in more than half
of the patients, (d) levels of blood components involved in immune
response were higher in vaccine responders, and (e) we built a
mathematical model that could predict pneumococcal vaccine response with
a 67\% probability.

Vaccination against pneumococcal disease is recommended in Spain
according to the Advisory Committee on Vaccines of the Spanish
Association of Pediatrics for all children under 5 years of age and
children of any age at risk of infection due to underlying diseases. In
Comunitat Valenciana, only the PCV13 is included in the childhood
immunization schedule since May 2015, and consequently, it is the most
found in our series\textsuperscript{19}.

Older patients (10-16 years) had lower anti-PCP IgG levels and higher
rates of previous IPD, possibly due to the poorer vaccination found in
our cohort (29.41\%). Nevertheless, in our study, no true pneumococcal
vaccine failure case was reported, despite recent series showing cases
of IPD by PCV13 serotypes in children who had been age-appropriately
vaccinated\textsuperscript{20}.

Vaccination response was inadequate or insufficient in more than half of
our patients regardless of the underlying disease, LHT being the group
with the lowest antibody titers in agreement with Fioredda et
al\textsuperscript{31}. Therefore, revaccination with a booster dose
should be indicated in patients with leukemia, especially after
chemotherapy\textsuperscript{32}.

Unexpectedly, patients with previous IPD did not present greater
anti-PCP IgG levels than patients without past IPD. A plausible
explanation for this finding is that naturally acquired immunity depends
on multiple factors as nasopharyngeal colonization or pneumococcal
capsular antigens and surface proteins, whereas naturally acquired
protection against IPD depends largely on antibodies against surface
protein antigens rather than anti-capsular ones\textsuperscript{21}.

In our cohort, all the patients presented a high risk of developing
pneumococcal disease and therefore, pneumococcal vaccination was
indicated. However, our data showed that 70.6\% of the patients lacked
some dose, especially of polysaccharide vaccine, which agrees with
similar series\textsuperscript{22,23}. Thus, further efforts should be
implemented to increase awareness and improve the rates of pneumococcal
vaccination coverage in these patients.

Vaccine response is the product of correct vaccination coverage and of
an adequate production of protective antibody titters. Therefore, even
if the patient receives the recommended vaccine doses, an adequate
immune system has to generate an antibody level high enough to prevent
future pneumococcal infections. In our study, 12 of the 25 properly
vaccinated patients did not reach an acceptable antibody level, as it
has been found in other series\textsuperscript{24,25}. This points out
the low effectiveness of vaccination in children with underlying
conditions affecting the immune system. In fact, we observed that
responders presented higher levels of immunoglobulins, complement
proteins and CD4/CD8 ratio than non-responders. These differences would
explain why these patients produced higher pneumococcal titers after
vaccination\textsuperscript{26}.

IgG4 deficiency is the most common subclass of immunodeficiency, and
15\% of normal children may present it. In our paper, we found that
correct immunization was achieved by children with higher levels of
IgG4. Our findings could explain Tian et al findings that link recurrent
respiratory infections --particularly pulmonary-- and bronchiectasis
with IgG4 serum deficiency\textsuperscript{27}.

In agreement with Avelino-Silva et al and Fuster et al, CD4/CD8 ratio
was one of the most important predictors of vaccine response, and a
lower ratio might be associated with impaired B cell
function\textsuperscript{28,29}. However, multiple elements such as
environmental, behavioral, nutritional, intrinsic and extrinsic host
factors influence vaccine response as well\textsuperscript{30}.

Clinical and laboratory correlations between low CD4 T lymphocyte counts
and C4 levels in patients with leukemia have been
published\textsuperscript{33}. The CD4 T lymphocyte is one of the main
effectors of vaccine response, especially for conjugate vaccines. Thus,
our LHT patients were prone to not responding to pneumococcal vaccines
because their CD4 T lymphocyte counts were under
500/mL\textsuperscript{34}. Interestingly, our LHT patients presented
the higher C4 levels, although within normal range and should not be
relevant\textsuperscript{35}.

Finally, we were able to build a mathematical model to predict
vaccination response with a 67\% accuracy based on age, IT, C3, C4 IgG3
and lymphocyte levels. The model we suggest is simple enough to allow
its use in clinical settings and to identify patients at higher risk of
vaccination failure.

IT and lymphocyte counts are critical factors in the prediction of
pneumococcal vaccine response\textsuperscript{36,37}. In our research,
IT was associated with lower response rates to pneumococcal vaccination
while absolute lymphocyte count predicted a protective
response\textsuperscript{38,39}. The correlation between age and
vaccination failure was also important, the mean age being higher in
patients with primary vaccine failure. A possible explanation to this
finding could be that younger patients were better vaccinated overall
than older patients as a result of recent vaccination guidelines and
risk awareness.

Although C3 plays a central role in the immune system, in our study high
levels of this component were negatively associated with pneumococcal
vaccine response. Recent studies suggest the close relationship between
elevated levels of C3 and diabetes or obesity, diseases in which the
role of the immune system has already been
demonstrated\textsuperscript{40}. On the other hand, elevated serum
levels of C4 and IgG3 were correlated with vaccine response, which was
consistent because both are essential components of the immune
system\textsuperscript{36}.

Limitations of the study include the relatively small number of patients
recruited and the heterogeneity in the type of pneumococcal vaccines
administered in the study population, which nonetheless, reflects the
current situation of our patients.

In conclusion, we have studied pneumococcal vaccination coverage and
vaccine response rates in children with underlying diseases. Our
findings highlight the conditions and clinical data that should be taken
into account to identify children at higher risk of vaccination failure.

\textbf{ACKNOWLEDGEMENTS}

The authors express their sincere gratitude to all subjects included in
the study.

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