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THE EFFECT OF OBESITY ON THE IMMUNE RESPONSE TO INFLUENZA VACCINATION IN THE ELDERLY

By

Nicole Buddenbaum

Senior Honors Thesis Department of Nutrition

University of North Carolina at Chapel Hill May 1st, 2020

Approved:

_____________________________ Melinda Beck, PhD (Advisor)

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ABSTRACT

Nicole Buddenbaum: The Effect of Obesity on the Immune Response to Influenza Vaccination in the Elderly

(Under the Direction of Melinda Beck)

Each year, influenza results in 3 to 5 million cases of severe illness and between 290,000 to 650,000 deaths globally.1 Elderly and obese individuals are at increased risk of infection, illness and death. Elderly individuals ( 65 years) exhibit a decline in immune function which

renders them more susceptible to influenza. Obese individuals (BMI  30 kg/m2) exhibit

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TABLE OF CONTENTS

LIST OF TABLES………...4

LIST OF FIGURES……….5

LIST OF ABBREVIATIONS AND SYMBOLS………6

CHAPTER I – BACKGROUND……….7

Introduction………...…...7

Influenza Virus and the Immune System……….8

Aging and the Influenza Vaccine………...11

Obesity and the Influenza Vaccine………12

Hypotheses and Aims………12

CHAPTER II – METHODS, RESULTS AND DISCUSSION……….13

Methods………..13

Results………22

Discussion………..29

Conclusion and Future Directions………...35

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LIST OF TABLES

Table 1 – Demographics of Elderly Non-obese and Obese Study Participants

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LIST OF FIGURES

Figure 1 – Test plate setup for Hemagglutination Inhibition Assay

Figure 2 – Pre- and Post- Influenza Vaccination Serum Antibody Levels to A/California/7/2009 (H1N1) in Elderly Non-obese and Elderly Obese Individuals

Figure 3 – Pre- and Post- Influenza Vaccination Serum Antibody Levels to A/Hong Kong/4801/2014 (H3N2) in Elderly Non-obese and Elderly Obese Individuals

Figure 4 – Percent Fold Increase in Antibody Levels for Elderly Non-obese and Elderly Obese Individuals by strain

Figure 5 – Correlation plots between Body Mass Index and Change in Titer (delta) to A/California/7/2009 (H1N1) in Elderly Individuals

Figure 6 – Correlation plots between Body Mass Index and Change in Titer (delta) to A/Hong Kong/4801/2014 (H3N2) in Elderly Individuals

Figure 7 – Correlation plots between Age and Change in Titer (delta) to A/California/7/2009 (H1N1) in Elderly Individuals

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LIST OF ABBREVIATIONS AND SYMBOLS

HA Hemagglutinin

NA Neuraminidase

A/California/7/2009 (H1N1) pdm09 2009 pandemic hemagglutinin 1 neuraminidase 1

H3N2 hemagglutinin 3 neuraminidase 2

HAI Hemagglutination inhibition assay

HAU Hemagglutination Units

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CHAPTER I – BACKGROUND Introduction

Influenza (flu) is a severe respiratory illness that affects millions worldwide. The World Health Organization estimates that, each year, influenza results in 3 to 5 million cases of severe illness and between 290,000 to 650,000 deaths globally.1 However, the elderly population, which consists of individuals >65 years of age, is disproportionally at risk; 70 to 85% of seasonal flu-related deaths and 50 to 70% of seasonal flu-flu-related hospitalizations occur in people 65 years and older.2 According to the Center for Disease Control and Prevention, age is a significant risk factor for influenza illness owing to a decline in immune function. This notion demands attention from a public health standpoint because recent improvements in healthcare and medicine have led to a steady increase in the percentage of the population worldwide who fall into the elderly category.3 As a result, hospitalization from influenza and death rates are expected to rise.

However, this phenomenon is not only attributable to the global shift in age distribution, but also to that of obesity prevalence, which has doubled worldwide since 1980.4 Obesity is defined as the over-accumulation of subcutaneous and/or abdominal adipose tissue. Excess adiposity results in a state of chronic, low-grade inflammation which negatively impacts immune function and host defense.5 With regards to influenza infection, obese hosts are more likely to exhibit impaired antiviral responses and poor recovery from disease compared with lean controls.6 As a result, the obese also constitute a high-risk population for influenza infection.7

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risk. Therefore, the purpose of this paper is to investigate the effect of obesity status on elderly immune response to influenza vaccination.

Influenza Virus and the Immune System

There are four types of influenza– A, B, C and D – all of which are spherical RNA viruses with surface glycoproteins hemagglutinin (HA) and neuraminidase (NA).9 The influenza A virus may be further classified based on the different subtypes of its surface proteins; there are 18 hemagglutinin subtypes (H1-H18) and 11 neuraminidase (N1-N11) subtypes. Upon entry into the body, the virus’ HA surface proteins recognize and bind to N-acetylneuraminic (sialic) acid residues on the surface of host cells.9 Following viral attachment, the virus is endocytosed and takes over the functions of the cell. The virus replicates, or copies itself, by using host ribosomes and amino acids to translate its RNA to produce more viral protein. The viral copies move to the cell membrane and are released out into the body where they go on to infect other cells.

The host immune system functions to defend against the influenza virus and clear it from the body in a process that involves both the innate and adaptive immune system.10 The innate immune system serves as the first line of defense against viral infection and consists of physical barriers, phagocytic cells, cytokines, and interferons. The adaptive immune system is the second line of defense and consists of B cells and T cells – specialized cells with antigen-specific memory that capture and neutralize pathogens. An important role of the adaptive immune system is establishing immunological memory, or the ability of the immune system to respond quickly and effectively to previously encountered pathogens.11

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and B lymphocytes of the adaptive immune system. Upon contact with a naïve adaptive

lymphocyte (that has not encountered the antigen and differentiated), the antigen presenting cell induces both humoral and cellular responses.12 The humoral response involves antibody

production against the pathogen by plasma cells differentiated from antigen-specific B cells, and the cellular response involves T cells.12 There are two main types of T cells, helper CD4+ T cells and cytotoxic CD8+ T cells. CD4+ T cells primarily amplify cytokine production and secretion and provide second signals for antibody production by B cells. CD8+ T cells primarily target infected cells through cytotoxicity. Memory T cells and B cells are also generated which are then able to rapidly and specifically respond to subsequent encounters with the influenza virus.12

Influenza virions constantly change and may do so via two separate processes: antigenic drift and antigenic shift. Antigenic drift refers to the process by which small changes or

mutations in the genes of the influenza virus lead to changes in the HA and NA the proteins of the virus.13 The small changes that occur from antigenic drift can accumulate over time and result in viruses that are antigenically different. These changes account for seasonal differences in the influenza virus. Influenza virons may also change due to antigenic shift, which is the mechanism responsible for occasional global outbreaks of influenza or pandemics. Antigenic shift occurs when there is an abrupt or major change in an influenza virus resulting in new HA and/or NA proteins and, consequently, a new influenza subtype in humans. Human influenza A and B viruses cause seasonal epidemics, and the influenza A subtypes H1N1 and H3N2 routinely circulate and cause illness.

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virus, the body recognizes viral surface glycoproteins as antigens, or foreign substances, and creates antibodies specific to those surface protein antigens. As a result, upon secondary

exposure to influenza, these circulating antibodies will bind to the virus to prevent entry into host cells. In general, individuals with an influenza A HA-specific antibody titer ≥ 40 detected by hemagglutination inhibition assay (HAI) are considered protected from subsequent infection, though this measure is controversial.14 In addition to producing antigen-specific antibodies, influenza vaccination stimulates T cell activation. As previously stated, the memory T cells that are generated (in response to either vaccination or infection) respond to subsequent encounters with the virus in a rapid and specific manner.

The flu vaccine must be updated each year to keep up with evolving influenza virions. When antigenic drift occurs, the immune system may not recognize the changes in the HA and NA surface protein antigens, which renders the antibodies less able to bind to the virus and prevent infection.13 When antigenic shift occurs, most individuals have little or no immunity against the new virus.13 Antigenic shift evades immunological memory, and since it takes time for the innate immune system to mount an immune response, the virus can propagate unchecked for several days, making infection much more severe. Fortunately, this process happens much less frequently than antigenic drift. As a result of both antigenic shift and antigenic drift,

individuals are encouraged to get vaccinated each year with the updated influenza vaccine, which contains the strains predicted to most likely affect individuals within a given season.

The influenza vaccine may be administered in the trivalent or quadrivalent form. The trivalent influenza vaccine (TIV) contains three inactivated forms of influenza, while the quadrivalent form contains four inactivated forms of influenza. Both the trivalent and

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virus, but the quadrivalent vaccine contains an additional influenza B virus. Vaccine-induced protection against the flu wanes after one year, which is another reason why it is strongly recommended that individuals be routinely vaccinated.

Aging and the Influenza Vaccine

A major role of the immune system is its ability to generate immunological memory, or recognize a specific antigen the body has previously encountered and initiate an immune

response in a quick and robust manner. This aspect of immunity is also the basis of vaccination, as previously described. However, aging is accompanied by a progressive decline in both cellular and humoral immunity. This decline in systemic immunity leads to a state of

“immunosenescence” in the elderly, a biological state accompanied by an increased prevalence of cancer, autoimmune and chronic diseases, poor responses to vaccination, and consequently, increased vulnerability to common infectious diseases, such as influenza.15 In fact, individuals 65 years and older are at a high risk of developing serious complications from the flu compared with young, healthy adults.16

Despite the gradual decline in immunity with age, vaccination remains effective in reducing influenza risk amongst elderly individuals. A 2011 study by Ademokun et al.

investigated immunization-induced influenza protection in healthy older adults, it was found that the percentages of both the young (18-49 years) and the old (65-89 years) individuals

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Obesity and the Influenza Vaccine

The obese also constitute a high-risk population for influenza infection. Obesity, defined as BMI ≥ 30 kg/m2, occurs through the deposition of excess lipids into adipose tissue. Excess adiposity leads to a chronic state of meta-inflammation with implications on humoral and

cellular immunity; obese hosts exhibit delayed antiviral responses to influenza infection and poor recovery from disease.19

Impairments in B cell and T cell activity in the obesogenic state have been hypothesized to increase susceptibility to influenza illness. Increased adiposity in obesity attracts B cells and increases B cell numbers.19 B cells of obese hosts have also been shown to exhibit alternations in secretomes following ex vivo stimualtion.19 Impaired T cell responses in obese adults have also been hypothesized for increased susceptibility to influenza; studies have reported deficiencies in the activation and function of CD4+ and CD8+ T cells from obese individuals following ex vivo stimulation with H1N1 virus.20

Obese individuals also exhibit diminished responses to the influenza vaccine.21 Our lab has demonstrated that, compared with vaccinated healthy-weight adults, vaccinated obese adults have twice the risk of influenza or influenza-like illness, despite equal serological response to vaccination.21 This suggests that impairments in cellular immunity may account for the higher observed risk of influenza in obese adults. However, the exact mechanism by which obesity impairs the immune response to influenza and influenza vaccine efficacy remains unclear. Hypotheses and Aims

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Aim 1: Determine influenza vaccination responses between elderly non-obese and elderly obese individuals. The hemagglutination inhibition assay (HAI) was performed on elderly pre- and post- influenza vaccination sera to determine if obesity influences antibody production, which served as a marker of humoral immunity. I hypothesized that elderly obese individuals would have lower HAI titer responses compared with the HAI titer responses from elderly non-obese individuals.

Aim 2: Determine antibody response varies by influenza viral strain type between elderly non-obese and elderly obese individuals. I hypothesized strain type would not have an effect on the vaccination responses from aim 1.

CHAPTER II – METHODS, RESULTS AND DISCUSSION Methods

Study Design

To investigate the effect of obesity on the elderly immune response to influenza

vaccination, a serological test, the hemagglutination inhibition (HAI) assay, was performed as a correlate of vaccine-induced immunity to the influenza virus. HAIs were performed on pre- and post- vaccination sera of elderly non-obese and elderly obese individuals to determine the level of antibodies against two influenza A strains, A/California/7/2009 (H1N1) and A/Hong

Kong/4801/2014 (H3N2). A total of 43 male and female individuals 65 years of age (elderly)

who received the 2016-2017 flu vaccine were selected for the study using the REDCap database. The demographics for these study individuals are shown in Table 1. Those with BMI < 30 kg/m2 were classified as non-obese, and those with BMI  30 kg/m2 were classified as obese. The

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with an age range of 65.9-81.2 years and a BMI range of 31.2-38.6 kg/m2. Pre- and post-

influenza vaccination sera were obtained for every study individual in each cohort and tested for antibodies against A/California/7/2009 (H1N1) and A/Hong Kong/4801/2014 (H3N2) – the two influenza A viruses included in the 2016-2017 flu vaccine. First, the HAI assay was performed to detect H1N1pdm09 antibodies against A/California/7/2009 (H1N1) for each individual’s pre- and post- vaccination sera, analyzed together on the same plate. Then, the HAI assay was

repeated in the same manner for each individual’s pre- and post- vaccination sera to detect H3N2 antibodies against A/Hong Kong/4801/2014 (H3N2). The HAI assay involved serial 2-fold dilutions, with an initial dilution of 1:10, for each of 2 sera (pre-vaccination and

post-vaccination) in 96-well v-bottom plates, followed by incubation with standardized concentrations of influenza A virus representing the vaccine composition from that study year. Turkey red blood cells (TRBCs) were added to wells and allowed to settle before hemagglutination was observed. The strain-specific HAI antibody titers for each individual were calculated as the reciprocal of the highest dilution of sera that inhibited hemagglutination.

Table 1. Demographics of Elderly Non-obese and Obese Study Participants

Subject

Number Sex Race Age BMI Obesity

08_3041 Female Caucasian 84.2 28.1 No

08_3061 Male Caucasian 66.2 25.9 No

08_3069 Female Caucasian 69.3 24.2 No

08_3081 Female Caucasian 71 26.3 No

08_3152 Male Caucasian 77.5 24.3 No

08_3162 Male Caucasian 77.9 21.6 No

08_3164 Female Caucasian 79.9 28.3 No

08_3185 Male Caucasian 74.9 22.9 No

08_3187 Female Caucasian 83.3 29.1 No

08_3188 Female Caucasian 84.4 21.8 No

08_3192 Male Caucasian 75.3 21.8 No

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08_3234 Male African American 74.9 25.4 No

08_3239 Female Caucasian 74.7 28.1 No

08_3245 Female Caucasian 73.1 24.3 No

08_3268 Female Caucasian 71.7 22.3 No

08_3284 Female Caucasian 84 24.3 No

08_3289 Male Caucasian 67.6 25 No

08_3089 Female Caucasian 68.8 21.1 No

08_3101 Female African American 75.2 28.2 No

08_3113 Male African American 67.2 25 No

08_3121 Male Caucasian 77.3 25 No

08_3128 Female Caucasian 74.6 23.4 No

08_3139 Female African American 69.3 26.4 No

08_3145 Female Caucasian 67.3 29 No

08_3052 Female Caucasian 65.9 32.8 Yes

08_3055 Female African American 66.5 32.9 Yes

08_2873 Male Caucasian 81.2 31.2 Yes

08_2972 Female African American 69.6 32.4 Yes

08_2829 Male Caucasian 71.6 31.2 Yes

08_2940 Male Caucasian 67.6 35.7 Yes

08_3119 Male Caucasian 69 35.8 Yes

08_3098 Female African American 67.8 33.5 Yes

08_3107 Male Caucasian 79.5 31 Yes

08_2863 Male Caucasian 72.7 31.4 Yes

08_3256 Female Caucasian 74.5 38.6 Yes

08_2996 Female Caucasian 73.9 31.6 Yes

08_2982 Male African American 76.8 33.3 Yes

08_3126 Female Caucasian 71.4 38.4 Yes

08_2980 Female Caucasian 73 35.7 Yes

08_3039 Female African American 69.8 33.6 Yes

Influenza A HAU Titer

Virus: 10-day-old embryonated chicken eggs were inoculated with either the

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0.5% RBC suspension: Turkey red blood cells (TRBCs) were stored at 4°C.

Approximately 2-4 mL of TRBCs were mixed with 10 mL sterile phosphate-buffered saline (PBS) using a serological pipette before 10 minutes of centrifugation at 4°C, 270 g and 5 mins deceleration. Cells were resuspended in 10 mL PBS and centrifuged (10 mins, 4ºC, 270 g, 5 deceleration) twice more, for a total of 3 washes. After the third centrifugation, the TRBC pellet size was estimated and DPBS was added to yield a 0.5% packed RBC suspension.

Titration of Influenza Virus: To obtain the hemagglutination units (HAU) of each influenza virus, 2-fold serial dilutions were performed prior to each HAI assay. The influenza A virus was thawed and stored on ice. To a 96-well v-bottom plate, 180 microliters of PBS were added to row A, columns 1-6. To columns 1-3, 20 microliters influenza A virus were added; to columns 4-6, 20 microliters sterile PBS were added. Sterile PBS (100 microliters) was also added to rows B-H, columns 1-6. For serial dilutions, row A was mixed, and 100 microliters were transferred to row B. Row B was mixed, then 100 microliters were transferred to row C. This process was repeated sequentially down the plate, changing tips for each row, until the last row (H). After mixing, the extra 100 microliters were discarded from row H. To a separate 96-well v-bottom plate, 50 microliters of dilutions were transferred from all 96-wells of the mixing plate to a testing plate. The 0.5% TRBC suspension (50 microliters) was added into each well and mixed thoroughly. The plate was gently tapped to mix, and incubated at room temperature for 30 minutes. After this time, the plate was read to obtain the HAU of the influenza virus; the highest dilution of virus causing complete agglutination was considered as endpoint. Once the viral titer was obtained, sterile PBS was added to the virus so that the diluted virus was 8 HAU.

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plate, 200 microliters sterile PBS were added to row A, columns 10-13 and 200 microliters diluted virus were added to tow A, columns 7-9. Sterile PBS (100 microliters) was added to all wells B through H, columns 7-12. Row A was mixed, then 100 microliters were transferred to row B. Row B was mixed, then 100 microliters were transferred to row C. This dilution process was repeated down the plate to the final row (H). After mixing, the extra 100 microliters were discarded from row H. To a separate 96-well v-bottom plate, 50 microliters of dilutions from all wells were transferred, starting at the bottom row and proceeding up the plate (lowest to highest concentration of virus). The 0.5% TRBC suspension (50 microliters) was added into each well and mixed thoroughly. The plate was gently tapped to mix and incubated for 30 minutes at room temperature to allow TRBCs to settle. The highest dilution of virus causing complete

agglutination was considered as endpoint; if the dilution fell between 1 HAU and 0.5HAU, the diluted virus was used for the HAI assay.

Influenza A HAI Assay

Receptor-destroying enzyme: Prior to HAI testing, all subject sera were incubated with receptor-destroying enzyme (RDE) in a 1:3 ratio at 37°C in a water bath for 18-20 hours. The RDE-treated sera were subsequently inactivated by heating in a 56°C water bath for 1 hour. Following inactivation, samples were cooled to room temperature before 6 parts physiological saline (0.85% NaCl in deionized H2O) were added, resulting in a final dilution of serum of 1:10. The RDE-treated and diluted sera were frozen at 40°C for at least 4 hours prior to use in the HAI assay.

Hemagglutination Inhibition Test: The diluted influenza A virus (adjusted to 8 HAU) was used in the HAI assay to determine serological antibody concentrations for each study

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an individual’s pre-vaccination serum adjacent to his post-vaccination serum for a given viral strain and on the same plate. Therefore, a total of two study subject sera were analyzed per HAI plate. The RDE-treated samples were thawed in a 37°C water bath and mixed by pipette. In a 96-well v-bottom plate, 25 microliters of PBS were added to all 96-wells, except A1-12 (Figure 1). To row A, columns 1 to 8, 50 microliters of individual subject sera were added. Subject sera were added in duplicates (columns 1 and 2 received the same sample, columns 3 and 4, etc.). To row A, column 9, 50 microliters of positive control serum were added. To row A, column 10, 50 microliters of negative control serum were added. To row A, column 11, 50 microliters of virus (8 HAU/50 microliters) were added. To row A, column 12, 50 microliters of PBS were added. All sera were serially diluted in 2-fold increments through row H, resulting in dilutions from 1:10 to 1:1280. After serial dilution of all sera, 25 microliters of diluted influenza virus were added to all wells in columns 1-10. To columns 11 and 12, 25 microliters of PBS (instead of virus) were added to all wells. After mixing, plates were incubated at room temperature for 15 minutes. Following incubation, 50 microliters of 0.5% TRBCs were added to all wells. Plates were tapped to mix, covered, and incubated for 20 minutes at room temperature. After, plates were tilted to observe wells for agglutination. Nonagglutinating cells were defined as those that contained a button of cells at the very bottom of the well, which could be seen running down the side of the well to the bottom edge of the well when the plate was tipped at a 45° angle.

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the sera and virus controls had to be followed: (1) values on HAI plates for the positive control serum had to be within 1 dilution of its known titer, (2) values on the HAI plate for the negative control serum had to equal 10 or 20, and (3) viral back-titers must have had values within 1 dilution of that observed on the test plate for the diluted virus.If at least one of these criteria were not satisfied, the assay was not accepted and no subject titers were analyzed. Table 2 and Table 3 show the pre- and post- vaccination titers for the elderly non-obese and elderly obese study individuals, respectively.

Table 2. Pre- and Post- Vaccination titers for Elderly Non-obese Study Individuals

Subject

Number Sex Age BMI

Pre-Vaccination Titer Post-Vaccination Titer

H1N1pdm09 H3N2 H1N1pdm09 H3N2

08_3041 Female 84.2 28.1 0 0 40 80

08_3061 Male 66.2 25.9 160 80 160 80

08_3069 Female 69.3 24.2 0 0 0 0

08_3081 Female 71 26.3 40 80 80 320

08_3152 Male 77.5 24.3 20 160 40 320

08_3162 Male 77.9 21.6 10 10 20 20

08_3164 Female 79.9 28.3 20 20 40 20

08_3185 Male 74.9 22.9 20 0 40 80

08_3187 Female 83.3 29.1 40 40 40 40

08_3188 Female 84.4 21.8 40 40 80 20

08_3192 Male 75.3 21.8 20 20 20 20

08_3202 Female 80.7 22.7 40 0 80 40

08_3210 Female 70 26.6 0 10 20 80

08_3222 Male 70.5 28.7 20 10 40 160

08_3234 Male 74.9 25.4 0 0 40 160

08_3239 Female 74.7 28.1 20 40 20 160

08_3245 Female 73.1 24.3 0 0 0 0

08_3268 Female 71.7 22.3 0 10 40 80

08_3284 Female 84 24.3 0 0 80 160

08_3289 Male 67.6 25 0 0 0 0

08_3089 Female 68.8 21.1 0 0 0 0

08_3101 Female 75.2 28.2 80 160 320 1280

08_3113 Male 67.2 25 160 40 320 320

08_3121 Male 77.3 25 320 40 1280 40

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08_3139 Female 69.3 26.4 40 40 40 640

08_3145 Female 67.3 29 0 40 0 80

Table 3. Pre- and Post- Vaccination Titers for Elderly Obese Study Individuals

Subject Number

Sex Age BMI Pre-Vaccination Titer Post-Vaccination Titer

H1N1pdm09 H3N2 H1N1pdm09 H3N2

08_3052 Female 65.9 32.8 80 40 80 40

08_3055 Female 66.5 32.9 20 160 160 1280

08_2873 Male 81.2 31.2 20 40 20 40

08_2972 Female 69.6 32.4 80 0 80 0

08_2829 Male 71.6 31.2 80 20 160 80

08_2940 Male 67.6 35.7 40 80 40 160

08_3119 Male 69 35.8 40 10 40 10

08_3098 Female 67.8 33.5 160 20 320 40

08_3107 Male 79.5 31 20 160 20 160

08_2863 Male 72.7 31.4 10 0 20 10

08_3256 Female 74.5 38.6 10 80 40 160

08_2996 Female 73.9 31.6 20 40 160 80

08_2982 Male 76.8 33.3 40 40 80 80

08_3126 Female 71.4 38.4 20 0 40 10

08_2980 Female 73 35.7 40 320 0 320

08_3039 Female 69.8 33.6 40 40 40 80

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A paired sample t-test was performed, for each viral, strain comparing mean pre- and post- vaccination titers of all elderly individuals within the non-obese and obese cohorts. An independent sample t-test was also performed, for each strain, to compare (1) the mean pre-vaccination titers of elderly non-obese and elderly obese individuals and (2) the average change (delta) in titer from pre- to post- influenza vaccination. Seroconversion, seroprotection, and the percentage of individuals who achieved a post-vaccination titer of 80 or above were compared between the non-obese and obese cohorts, for each strain. Seroconversion was defined as a 4-fold rise in antibody titer or greater, and seroprotection was defined as a titer of 40 or above. Finally, correlation plots were made to investigate the relationship between (1) BMI and change in titer from pre- to post- vaccination in all elderly subjects, and (2) age and change in titer from pre- to post- vaccination in all elderly subjects.

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Figure 2. Pre- and Post- Influenza Vaccination Serum Antibody Levels to A/California/7/2009 (H1N1) in Elderly Non-obese and Elderly Obese Individuals

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pre- and post-vaccination H1N1pdm09 antibodies; only a significant increase was exhibited for the non-obese elderly cohort. However, the average increase in titer was not statistically different between obese and non-obese elderly individuals (Figure 2E).

Figure 3. Pre- and Post- Influenza Vaccination Serum Antibody Levels to A/Hong Kong/4801/2014 (H3N2) in Elderly Non-obese and Elderly Obese Individuals

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titers for the A/Hong Kong/4801/2014 (H3N2) strain (Figure 3C). Figure 3D compares obese and non-obese elderly pre- and post-vaccination H3N2 antibodies; a significant increase in HAI titer was exhibited by both the obese and non-obese elderly cohorts. The average increase in titer between the two groups (Figure 2E) was not statistically different.

Figure 4. Percent Fold Increase in Antibody Levels for Elderly Non-obese and Elderly Obese Individuals by strain

Figure 4 shows that non-obese and obese elderly individuals exhibited no significant difference in seroconversion, seroprotection, or the percentage of individuals who reached a post-titer of 80 and above for the A/California/7/2009 (H1N1) strain. For the A/Hong

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Figure 5. Correlation plots between Body Mass Index and Change in Titer (delta) to A/California/7/2009 (H1N1) in Elderly Individuals

Correlation plots were made between body mass index (BMI) and change in titer (delta) to the A/California/07/2009 (H1N1) strain for all elderly study subjects (Figure 5). There is a nonsignificant (p=0.15), negative and weak (R=-0.24) association between BMI and

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Figure 6. Correlation plots between Body Mass Index and Change in Titer (delta) to A/Hong Kong/4801/2014 (H3N2) in Elderly Individuals

Correlation plots were made between body mass index (BMI) and change in titer (delta) to the A/Hong Kong/4801/2014 (H3N2) for all elderly study subjects (Figure 6). Figure 6A shows a nonsignificant (p=0.82), positive and very weak (R=0.038) correlation between elderly BMI and change in antibody levels to the A/Hong Kong/14 strain following influenza

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(H3N2) strain following influenza vaccination. When only obese elderly individuals are analyzed (Figure 6C), a similar nonsignificant (p=0.22), positive and weak (R=0.35) correlation between BMI and change in antibody levels to the A/Hong Kong/14 strain following influenza

vaccination is obtained.

Figure 7. Correlation plots between Age and Change in Titer (delta) to A/California/07/2009 (H1N1) in Elderly Individuals

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0.46) correlation between age and change in antibody levels to the A/California/7/2009 (H1N1) strain following influenza vaccination when non-obese and obese elderly subjects are pooled. Figure 7B also shows a statistically significant (p=0.75), positive and moderate (R=0.55) correlation between an elderly individual’s age and change in antibody levels to the

A/California/07/2009 (H1N1) strain following influenza vaccination when only the non-obese elderly individuals are analyzed. However, Figure 7C shows a nonsignificant (p=0.47), positive and weak (R=0.22) correlation when only the obese elderly individuals are analyzed.

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The average change in titer (delta) to the A/Hong Kong/4801/2014 (H3N2) from pre- to post- influenza vaccination was graphed as a function of age for all study individuals (Figure 8). Figure 8A shows a nonsignificant (p=0.98), positive and very weak (R=0.0043) correlation between an elderly individual’s age and change in antibody levels to A/Hong Kong/4801/2014 (H3N2) following influenza vaccination when both obese and non-obese elderly study subjects are pooled. Figure 8B shows a nonsignificant (p=0.75), negative and weak (R=-0.063)

correlation and Figure 8C shows a nonsignificant (p=-.95), negative and very weak (R=-0.017) correlation between an elderly individual’s age and change in antibody levels to the A/Hong Kong/4801/2014 (H3N2) when either non-obese or obese elderly study subjects are analyzed, respectively.

Discussion

Influenza is a highly contagious and severe respiratory disease that substantially burdens human health and claims the lives of millions each year. The elderly and obese populations, however, are particularly at risk for influenza due to impaired immunity. The anticipated increase in the proportion of the global population that constitutes both elderly and obese therefore necessitates investigation into the extent to which aging and obesity, together, impact influenza risk and the immune response to influenza vaccination.

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effect, if any, of the viral strain type on this response. All elderly individuals under study were vaccinated with the 2016-2017 influenza vaccine containing the A/California/7/2009 (H1N1) pdm09, A/Hong Kong/4801/2014 (H3N2), and B/Brisbane/60/2008 viral strains. Due to the greater immunogenicity of the A strains, serum samples were tested for antibodies against the A/ California/7/2009 (H1N1) pdm09 and A/Hong Kong/4801/2014 (H3N2) strains.

Overall, the results fail to suggest a statistically significant effect of obesity on influenza vaccination responses in the elderly study subjects. Figure 2 shows the pre- and post-

vaccination titers to the A/California/7/2009 (H1N1) pdm09 strain for the obese and non-obese elderly subjects. Although the data indicate that the non-obese cohort exhibited a significant increase in H1N1pdm09 antibodies following vaccination whereas the obese cohort did not, this phenomenon is likely a result of the strain type investigated.

Since the 2009 flu pandemic, the World Health Organization (WHO) recommended integration of the swine-based virus A/California/07/2009 (H1N1) strain in yearly vaccinations.22 As a result, this particular strain has been incorporated into the influenza vaccine in the Northern and Southern Hemispheres each year since 2010, and beyond the 2016-2017 study year that individuals were selected from. Depending on frequency of flu vaccination, study participants could have been exposed to and developed antibodies against the A/California/07/2009 (H1N1) strain due to being vaccinated 6 times in sequential years prior to our testing. The high pre-vaccination titer levels for the obese suggests that the elderly obese individuals may have previously received influenza vaccination, or had developed an influenza infection, more than the elderly non-obese individuals before the start of the study.

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obese subjects often present with other health complications and thus require frequent visits to their healthcare providers. This renders them more likely to receive medical attention and consequently, get vaccinated. Alternatively, individuals in the obese cohort may have contracted the flu in the past, which could also explain their high titers at baseline. Taken together, previous encounter of influenza vaccination or infection may contribute to the high pre-vaccination titer observed in the elderly obese individuals, which was found to be significantly different from the pre-vaccination titer of the elderly non-obese individuals. The fact that this elderly obese cohort did not exhibit a significant increase in titer following vaccination suggests that post-vaccination antibody production may increase only up to a certain threshold. Therefore, the fact that elderly obese individuals did not exhibit a significant increase in titer less likely represents an effect of obesity on impaired humoral response but rather an effect of pre-vaccination titer. In other words, since elderly obese individuals already had high pre-titer levels, they experienced no significant increase in H1N1pdm09 antibody production following influenza vaccination compared with the elderly non-obese individuals.

Figure 2E further supports the notion that obesity status does not impair vaccination response in the elderly because the overall change (delta) in titer following influenza vaccination was not statistically different between the non-obese and obese cohorts. Since the average change is not statistically different between the two demographics, the data do not suggest that elderly obese individuals exhibit a lesser antibody response to influenza vaccination.

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Kong/4801/2014 (H3N2) strain was incorporated into the influenza vaccine for the first time in the 2016-2017 flu season corresponding to the year study samples were taken from. Since neither cohort had prior vaccine exposure to the strain, pre-vaccination titers in both groups were not statistically different, and therefore, all individuals exhibited a significant increase in H3N2 antibodies following vaccination. That is, all elderly individuals, regardless of obesity status, experienced an increase in post-influenza vaccination titer. To add, there was no statistically significant difference between the average increase (delta) in titer to the A/Hong

Kong/4801/2014 (H3N2) strain among elderly non-obese and elderly obese individuals (Figure 3E). These findings do not suggest an effect of obesity status on antibody production following influenza vaccination.

To further quantify the effect of obesity on elderly influenza vaccination response, elderly obese and non-obese seroconversion and seroprotection were compared, as well as the percentage of individuals who reached a post-titer of 80 and above (Figure 4). Seroconversion and seroprotection are correlates of immunity in vaccinology.23 Seroconversion is defined as a 4-fold rise in antibody titer or greater, and seroprotection is defined as a titer of 40 or above. The data show that elderly non-obese and elderly obese individuals did not statistically differ in seroprotection or the percentage of individuals who reached a post-titer of 80 and above, regardless of the influenza strain tested. Since a titer of 40 (seroprotection) means that 50% of the population with this titer will be protected from disease, and both the elderly non-obese and obese individuals reached this threshold, the data fail to suggest a significant effect of obesity status on elderly influenza vaccination response.

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investigated. However, this significance is most likely a result of the wide variation in the data as opposed to a biological phenomenon between obese and non-obese elderly individuals.

Seroconversion attempts to quantify the magnitude of change for titer levels following vaccination. As previously discussed, elderly obese and non-obese individuals showed a nonsignificant difference between change in antibody levels to the A/Hong Kong/4801/2014 (H3N2) strain, but there was a high degree of variability within that dataset. To add, there was no significant difference between elderly obese and non-obese seroprotection or the percentage who reached a titer of 80 and above for that strain. This, taken together with the data from Figure 3C which suggest no significant difference in H3N2 antibody production following vaccination, suggests that the difference in seroconversion observed for the A/Hong Kong/4801/2014 (H3N2) strain is a result of the variability in the data, and not an effect of obesity on elderly vaccination response.

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To investigate the trend in aging and influenza vaccination response, titer change

following influenza vaccination was graphed as a function of age for all elderly study individuals and against each A strain (Figure 7 and Figure 8). There is a significant association between age and H1N1pdm09 titer change when obese and non-obese elderly individuals are pooled in Figure 7A, which implies that as elderly individuals continue to age, their antibody titer

increases following flu vaccination. However, it is possible that the non-obese elderly individuals are driving this response because on average, non-obese individuals live longer. It is also well-established that age correlates with a decreased response to the flu vaccine.25 Therefore, separate correlation plots were made separating elderly obese and non-obese study subjects.

There is a significant, positive and moderate association between H1N1pdm09 titer change and age for elderly non-obese subjects. This may reflect the fact that the elderly individuals who are not obese have better health status and did not previously contract the influenza virus. As a result, when vaccinated, they experienced a significant increase in antibody levels to A/California/07/2009 (H1N1), which is why their antibody change appears to increase with age. However, there is a nonsignificant, positive and very weak correlation between H1N1pdm09 titer change and age amongst elderly obese subjects. This may reflect the fact that elderly persons who are obese at all ages present with greater health complications, and

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overall would reflect a negative correlation between age and titer change following influenza vaccination.

The average change in titer from pre- to post- influenza vaccination against the A/Hong King/4801/2014 (H3N2) strain was also plotted as a function of age for all elderly study subjects. The nonsignificant trend in Figure 8A indicates that after reaching a certain age (e.g. 65 years), titer levels neither increase nor decrease regardless of obesity status. The trend is nonsignificant with no appreciable association between age and H3N2 antibody production amongst elderly non-obese individuals. Similarly, the trend is nonsignificant with no statistically significant association between the two variables amongst elderly obese individuals. Since the correlation plots for both groups are comparable, the data do not suggest an effect of obesity status on vaccination response. This similarity is likely attributable to strain type; since neither the elderly obese nor non-obese individuals could have been exposed to the H3N2 viral strain prior to the study, neither group could have made antibodies against it. Importantly, since these graphs do not indicate a decline in antibody response with age, especially after vaccination with a new strain, this suggests that all elderly individuals, regardless of obesity status, should continue to get vaccinated.

Conclusion and Future Directions

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A/California/07/2009 (H1N1) and A/Hong Kong/4801/2014 (H3N2) strains. Further, Figure 4 indicates that seroprotection and the percentage of individuals who reached a titer of 80 or above were not significantly different between elderly obese and non-obese individuals, regardless of strain. Figure 5 and Figure 6 suggests that, as BMI increases, there is no significant effect on elderly vaccination response. Finally, Figure 7 and Figure 8 show little to no correlation between titer and increasing age in the elderly, regardless of obesity status.

Though the data from this study do not suggest a difference in vaccination response for elderly obese and elderly non-obese individuals, previous data demonstrated obese individuals compared with non-obese controls, are more susceptible to influenza.20 In this study, antibody responses were measured in serum collected from elderly individuals one month

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