Administration of thimerosal-containing vaccines to infant rhesus macaques does not result in autism-like behavior or neuropathology

Edited by Matthew State, University of California, San Francisco, CA, and accepted by the Editorial Board August 9, 2015 (received for review January 15, 2015)
September 28, 2015
112 (40) 12498-12503

Significance

Autism is a childhood neurodevelopmental disorder affecting approximately 1 in 70 children in the United States. Some parents believe that thimerosal-containing vaccines and/or the measles, mumps, rubella (MMR) vaccine are involved in the etiology of autism. Here we gave nonhuman primate infants similar vaccines given to human infants to determine whether the animals exhibited behavioral and/or neuropathological changes characteristic of autism. No behavioral changes were observed in the vaccinated animals, nor were there neuropathological changes in the cerebellum, hippocampus, or amygdala. This study does not support the hypothesis that thimerosal-containing vaccines and/or the MMR vaccine play a role in the etiology of autism.

Abstract

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder. Some anecdotal reports suggest that ASD is related to exposure to ethyl mercury, in the form of the vaccine preservative, thimerosal, and/or receiving the measles, mumps, rubella (MMR) vaccine. Using infant rhesus macaques receiving thimerosal-containing vaccines (TCVs) following the recommended pediatric vaccine schedules from the 1990s and 2008, we examined behavior, and neuropathology in three brain regions found to exhibit neuropathology in postmortem ASD brains. No neuronal cellular or protein changes in the cerebellum, hippocampus, or amygdala were observed in animals following the 1990s or 2008 vaccine schedules. Analysis of social behavior in juvenile animals indicated that there were no significant differences in negative behaviors between animals in the control and experimental groups. These data indicate that administration of TCVs and/or the MMR vaccine to rhesus macaques does not result in neuropathological abnormalities, or aberrant behaviors, like those observed in ASD.
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder presenting in early childhood with a current prevalence ranging from 0.7% to 2.64% in the United States (1). ASD is defined by the presence of marked social deficits, specific language abnormalities, and stereotyped repetitive patterns of behavior (2). Genetic and environmental factors have been found to play a role in the disorder (3, 4). The neuropathology of autism is now beginning to be understood; however, there is still much to be learned. Thus far, the major neuropathological changes observed in autism are changes in neuronal size in the limbic system; decreased numbers of Purkinje cells in the cerebellum; abnormalities in the brainstem, neocortex, amygdala, and hippocampus; features of cortical dysgenesis or migration disturbances; and alterations in GABAergic and cholinergic systems [see Gadad et al. (3) and Amaral (5) for reviews]. In many autism studies, comorbid conditions such as seizure disorders or intellectual disabilities contribute to the heterogeneity of the neuropathology.
An association between exposure to thimerosal-containing vaccines (TCVs) and developmental abnormalities has been debated since 1999 when the US Food and Drug Administration determined that children receiving multiple TCVs at a young age were at risk for exceeding the Environmental Protection Agency’s safe exposure limits for methylmercury (MeHg). Results from an Institute of Medicine (IOM) review on the safety of childhood vaccines found that there was not sufficient evidence to render an opinion on the relationship between exposure to TCVs or the measles, mumps, rubella (MMR) vaccine and developmental disorders in children (IOM 2001) (6). The IOM review did, however, note the possibility of such a relationship and recommended further studies be conducted. A more recent second review of TCVs and autism (IOM 2004) (7) came to the same conclusion reached earlier: that there was no epidemiological data to support a relationship between TCVs and childhood developmental disorders. Several epidemiological studies sought to determine whether TCVs resulted in neurodevelopmental disorders including autism; however, both nonsignificant and significant associations have been reported (812). Significant associations have been reported by Thompson et al. (11), who investigated the association between TCVs and immune globulins early in life and neuropsychological outcomes in children at 7–10 y of age. The data included the evaluation of 1,047 children and their biological mothers and 24 neuropsychological tests. The only variable that was statistically significant was tics; children who were exposed to higher doses of thimerosal were more likely to exhibit tics. In a follow-up study by Barile et al. (12) examining a subset of the data from Thompson et al. (11), they found a significant association between thimerosal dosage and tics, but only in boys. They found no statistically significant associations between thimerosal exposure from vaccines early in life and six of the seven neuropsychological constructs examined.
Concern regarding the safety of childhood vaccines has had a major impact on immunization rates (1316). It is of great importance to determine whether TCVs play a significant role in altering brain development and/or behaviors that mimic changes observed in autism. The present study provides a comprehensive analysis of the influence of TCVs on the brain and behavior in a nonhuman primate model. The study includes 79 rhesus macaques in six groups (n = 12–16 per group): (i) Control, a control group given saline injections; (ii) 1990s Pediatric, replicating the pediatric vaccination schedule used for infants in the 1990s that included several TCVs; (iii) 1990s Primate, replicating the pediatric vaccination schedule used in the 1990s but accelerated fourfold representing the faster development of infant macaques; (iv) TCVs, only TCVs and no MMR; (v) MMR, only the MMR vaccine; and (vi) 2008, the expanded pediatric schedule used in 2008 (and very similar to that used today, which also includes a prenatal influenza vaccine; Table 1). For neuropathology, only animals in the 1990s and 2008 vaccine groups were studied because the 1990s schedule had the highest thimerosal exposure, and the 2008 schedule had the greatest number of different vaccines and is very similar to the vaccine schedule currently recommended for US infants. Analyses of early learning and cognition, from birth to 12 mo of age, in the same animals used in this study was recently reported by Curtis et al. (17).
Table 1.
Vaccination schedules used for the six groups of animals
GroupNVaccines administered
Control16None, all saline placebos
1990s Pediatric12Vaccine regimen as recommended in the 1990s
1990s Primate12Vaccine regimen as recommended in the 1990s accelerated fourfold
TCV’s12All TCVs and saline placebo for MMR
MMR15MMR only, all others replaced with saline placebo
200812Vaccine regimen recommended in 2008

Results

Social Behavior.

Overall means and SDs for duration and frequency of social and nonsocial behaviors scored for all animals are shown in Table 2. A description of the specific behaviors measured in this study is given in Table S1. The duration and frequency of negative behaviors (e.g., Stereotypy, Rock-huddle-self-clasp, Fear-disturbed, and Withdrawal) by animals in all groups across the entire study period was very low. Behaviors that had either a significant time main effect or a time × group interaction are shown in Fig. 1 (Social: Positive Behaviors; Nonsocial: Passive Behavior; and Nonsocial: Positive Behavior). The Nonsocial Explore behavior was the most frequent of the nine behaviors measured and presented the only instance of a significant effect involving group: there was a significant time × group interaction [F(5, 393) = 4.17, P = 0.004]. Follow-up contrasts indicated that the Control animals exhibited significantly more Nonsocial Explore behavior at the beginning of social living compared with the 1990s Primate [t(393) = 3.61, P < 0.001], the 1990s Pediatric [t(393) = −7.46, P < . 001], the MMR [t(393) = −2.72, P = 0.011], and the TCV [t(393) = −2.48, P = 0.017] groups (Fig. 1). However, there were no significant differences in any behavior measured between the control and experimental groups after 6 mo of social living (at ∼18 mo of age).
Table 2.
Duration and frequency (mean ± SD) of social and nonsocial behaviors scored for all 79 animals
BehaviorSocialNonsocial
Duration (SD)*Frequency (SD)Duration (SD)*Frequency (SD)
Passive7.77 (6.95)0.46 (0.25)1.67 (2.85)0.02 (0.03)
Explore3.51 (3.30)0.45 (0.19)164.89 (15.18)17.29 (2.74)
Play14.95 (5.37)3.67 (1.16)4.01 (2.02)2.56 (0.97)
Sex0.95 (1.00)0.16 (0.14)0.00 (0.00)0.00 (0.00)
Aggression0.03 (0.07)0.01 (0.02)0.02 (0.12)0.00 (0.01)
Withdrawal0.04 (0.14)0.01 (0.03)0.00 (0.00)0.00 (0.00)
Fear-disturbed0.29 (0.53)0.06 (0.09)0.34 (0.72)0.06 (0.11)
Rock-huddle-self-clasp0.02 (0.15)0.00 (0.01)0.00 (0.00)0.00 (0.00)
Stereotypy0.00 (0.00)0.00 (0.00)0.27 (0.72)0.03 (0.07)
Scoring was collected during 5-min focal periods collected 5 d/wk from ∼12 to 18 mo of age. Additional behaviors scored but not included in the analyses included eating, drinking, scratching, and self-grooming.
*
Duration reported in seconds. Frequency reported as number of events per session.
Fig. 1.
Analysis of behavioral data. Fitted values from analytical models of social and nonsocial behavior for groups from age 12 to 18 mo, back-transformed with antilog. Durations of positive behaviors (play, sex, and aggression) were summed for each animal. Only behaviors that showed either a significant time main effect or a time × group interaction are shown: (A) Social: Positive Behavior, (B) Nonsocial: Explore Behavior, (C) Nonsocial: Passive Behavior, and (D) Nonsocial: Positive Behavior. Nonsocial: Explore Behavior demonstrated the only significant time × group effect, and this was only significant at the beginning of social living. Duration of behaviors is shown in seconds.
Table S1.
Description of social and nonsocial behavioral categories scored for all animals
Behavior*Description of behaviors
PassiveNo intense interaction with other animals, self, or objects. Can include a slow visual scanning component, social contact such as huddling, or proximity within one foot, and occurs without locomotion.
ExploreVisual and/or tactual inspection of other animals, self, or objects, with or without locomotion.
PlayBehaviors with greater physical intensity than explore, involving “ears back-mouth puckering” expression, open mouth without teeth exposure or ears back, chasing, wrestling, bouncing, running or jumping, rolling, biting without injury, or ‘tug-of-war’ with an object.
SexPresenting rear area, inspection of genitalia, masturbation, with thrusting toward another animal or tester, mounting and thrusting an animal or object.
Aggression‘Stiff’ stance, piloerection, open-mouth threat, back, rolling and hitting with or without injury.
WithdrawalRetreat from an animal or object creating increased distance by locomotion, but with no fear behaviors.
Fear-disturbedFear display involving submissive posture, retraction of lips, cooing, screeching, convulsive jerking, or three successive hoots, with or without withdrawal or locomotion.
Rock-huddle-self-claspStrong clasping/grasping of another monkey without play behavior, or self-clasping with arms, legs, hands, or feet, without locomotion and no active inspection of own or other’s body.
StereotypyRepetitive body movements, with or without locomotion, requiring three or more consecutive, repetitive movements.
Reproduced with permission from ref. 17.
*
Behaviors can be either interactive with other animals (social behavior) or individual behaviors not involving any other animal (nonsocial behavior).

Brain.

The neuroanatomical analyses were first performed in brains from the 1990s Primate and 2008 groups, as animals in these groups received the highest amount of EtHg exposure (1990s Primate) or the most extensive vaccine exposure (2008). Because no neuronal differences were found in either of these vaccine groups compared with the control group, no additional vaccine groups were fully studied.

Cerebellum.

Abnormalities in the cerebellum have been reported in postmortem ASD brains (18, 19). Both histological and neurochemical analyses were performed on the cerebellar tissues in the present study.
Cerebellar volume and Purkinje cell number.
Stereological methods were used to estimate the total number of Purkinje cells (Fig. 2) in one hemisphere. There were an average of ∼800,000 cells in one hemisphere, with a density of 270 cell/mm3 and an overall volume of ∼3,000 mm3. No difference in cell number, density, or cerebellar hemisphere volume was observed in the 1990s Primate and 2008 groups compared with the Control group. We also examined Purkinje cell number in some of the animals in the TCV and MMR groups, and they were similar to that of the Control group (Table S2).
Fig. 2.
Cerebellar Purkinje cells. Purkinje cells are illustrated in sections stained with Cresyl violet (A and B) and calbindin-D28k/neutral red (C–E). C illustrates two regions, shown at higher power in D and E, illustrating that not all Purkinje cells stain positive for calbindin (D vs. E). There was no difference in the Purkinje cell number, cell density, or cerebellar hemisphere volume among the Control, 1990s Primate and 2008 groups (F–H). Sample size: n = 16 for Control; n = 12 for 1990s Primate; n = 8 for 2008. [Scale bars, (A) 50 µm; (B) 10 µm; (C) 2.2 mm; (D) 200 µm; and (E) 200 µm.]
Table S2.
Purkinje cell number (mean ± SEM) for five groups of animals
GroupMean ± SEM
Control (n = 16)795,754 ± 10,544
1990s Primate (n = 12)777,423 ± 6,560
2008 (n = 8)800,267 ± 14,811
TCV (n = 5)824,977 ± 18,129
MMR (n = 5)787,866 ± 8,422
ANOVA indicated that there was no difference among the groups (F = 1.68, P = 0.172).
Purkinje cell size.
Cell size (area) was measured in both Nissl-stained sections and in calbindin-immunostained sections. The calbindin-containing Purkinje cells were markedly larger than the calbindin-negative/Nissl-positive cells [Control mean ± SD (μm2) = 488.5 ± 7.9 and 273.1 ± 7.7; n = 8], but there was no difference in cell size between the Control and the 1990s Primate group for either calbindin-positive cells or Nissl-positive cells, respectively (Table S3).
Table S3.
Purkinje cell size measured in tissue stained for both Nissl and calbindin (n = 8/group)
Control1990s Primate
NisslCalbindinNisslCalbindin
273.1 ± 7.7488.5 ± 7.9279.2 ± 15.4502.1 ± 9.8
Data represent mean ± SEM (μm2).
Cerebellar proteins.
Western blots were run to measure the levels of Purkinje cell-related proteins—calbindin and GAD-67—and glial proteins—Iba1 (microglial marker) and GFAP (astrocyte marker) (Fig. 3). There were no differences in the protein levels in the 1990s Primate or 2008 groups compared with the Control group (n = 8/group). Because different regions of the cerebellum were used for the protein assays, it was important to ensure that the results reflect “whole cerebellum differences.” Therefore, we measured levels of the four proteins in five different cerebellar regions and found that all of the regions had similar levels of these proteins (Fig. S1).
Fig. 3.
Western blots of cerebellar proteins. (A) No differences were found in protein amounts for control, 1990s Primate, and 2008 groups. (B) Quantification of optical density values. Sample size: n = 8 for each of the three groups.
Fig. S1.
Western blots of cerebellum proteins. Proteins were measured from five different regions of the cerebellum in one brain. The density of calbindin, GFAP, Iba1, and GAD-67 is similar in all cerebellar regions.

Hippocampus.

The CA1 neurons in the hippocampus have been reported to be reduced in size in postmortem brains from children with autism (18).
CA1 cell size.
Cell size (area) was measured in Nissl-stained sections at a rostral (section 100), middle (section 200), and a caudal (section 300) level of the CA1 region (Fig. 4). Approximately 250–450 cells were measured per animal, each with a clear nucleolus at the three levels of the nucleus. There was no significant reduction in cell area for the 1990s Primate group vs. Control group or for the 2008 group vs. Control group.
Fig. 4.
CA1 cells in the hippocampus. (A) Location of the CA1 region. (B) Neurons at a higher magnification. Red arrows point to cells with a visible nucleolus. (Inset, Right) High magnification view of two neurons, one with a visible nucleolus. (C) Cell size data for Control (n = 16), 1990s Primate (n = 12), and 2008 (n = 8) groups. [Scale bars, (A) 1 mm and (B) 25 µm.]
Neurogenesis.
We sought to determine whether the birth of new dentate granule neurons was altered by the 1990s Primate vaccination schedule. Using doublecortin immunostaining, we counted the number of these neurons in five rostral-caudal sections/brain in the Controls and animals from the 1990s Primate group. There was no difference in the total number of cells per brain between the two groups (mean ± SEM for control brains: 4,180 ± 308 neurons, and 3,983 ± 368 neurons in the 1990s Primate group; n = 5/group; Fig. S2).
Fig. S2.
Newborn cells in the granule cell layer. The new dentate gyrus neurons are illustrated in a doublecortin (black cells) immunostained section. This section was counterstained with neutral red. (A–C) Black-labeled doublecortin cells at higher magnifications.
Dentate gyrus area.
We examined the area of the granule cell layer of the dentate gyrus in the Control, 1990s Primate, and 2008 groups at eight rostral-caudal levels through the structure (Fig. 5). A two-way ANOVA was run to compare area of the dentate gyrus in these three groups across the rostral-caudal extent of the nucleus. There was no significant difference in area among the three groups (P = 0.7565); however, as expected, there was a significant effect for rostral-caudal level (P < 0.0001).
Fig. 5.
The dentate gyrus. The size and shape of the dentate gyrus changes from rostral (A) to caudal (C). Illustrations were taken from sections 191, 251, and 331 (A–C). (D) Area of the dentate gyrus in the three groups of animals (Control, n = 12; 1990s Primate, n = 12; and 2008, n = 8). No group difference was found (ANOVA, P = 0.7565). [Scale bars, (A–C) 650 µm.]

Amygdala.

Abnormalities have been reported for the amygdala in ASD subjects (20). We measured the volume of the entire amygdala and of the lateral nucleus of the amygdala and the cell size and cell number for the lateral nucleus (Fig. 6). The volume of the amygdala was not significantly different in animals receiving either the 1990s Primate (n = 12) or 2008 (n = 8) vaccination schedules compared with the Controls (n = 16). In these same animals, we measured the volume and number of neurons in the lateral nucleus of the amygdala, and there was no difference among the three groups. Finally, the cell size in the lateral nucleus was not changed by either the 1990s Primate or 2008 vaccination schedules.
Fig. 6.
The amygdala was studied in three groups of animals: Control, 1990s Primate, and 2008. (A–F) Sections through the rostral-caudal extent of the amygdala stained for Nissl substance. Outlines are provided for the amygdala borders, and the lateral nucleus of the amygdala (L). The amygdala volume (G), lateral nucleus of the amygdala volume (H), lateral nucleus of the amygdala cell area (I), and lateral nucleus of the amygdala cell number (J) did not change in the 1990s Primate, 2008, and Control groups. Sample size: n = 12 for Control; n = 12 for 1990s Primate; n = 8 for 2008. (Scale bar, 2 mm).

Discussion

The association between exposure to TCVs and developmental outcomes has been debated since 1999 when the US Food and Drug Administration determined that children who received multiple TCVs at a young age were at risk for exceeding the Environmental Protection Agency’s safe exposure limits for MeHg. However, the safety limits for EtHg, found in TCVs, has not been extensively tested for its relationship with childhood developmental disorders.
In postmortem brains of subjects with ASD, reductions in the number of cerebellar Purkinje cells (18, 19) and amygdala lateral nucleus cells (20), and reductions in the cell size of CA1 hippocampal cell (18) have been reported. In the present study, infant male rhesus macaques received TCVs following the pediatric schedule from the 1990s (e.g., hepatitis B vaccine, diphtheria, tetanus, acellular pertussis vaccine, Haemophilus influenza B vaccine, measles, mumps, rubella vaccine) and the expanded 2008 schedule, and were euthanized at ∼18 mo of age. We examined cerebellar, amygdalar, and hippocampus neurons (n = 8–16/group), as these brain regions have been reported to be abnormal in postmortem brains from subjects with autism. There were no significant differences in Purkinje cell number or cell size, cerebellar volume, CA1 cell size, dentate gyrus volume, hippocampal neurogenesis, or lateral nucleus of the amygdala volume/cell number in animals in the 1990s Primate or 2008 groups compared with control animals. Our data do not support a role for TCVs in the neuropathology of ASD. A similar study examining the effects of TCVs on mouse neuropathology also reported normal hippocampal architecture with no changes in volume or numbers of neurons in the CA1 region or dentate gyrus (21).
There are limited studies on whether low-dose thimerosal via vaccination causes behavioral symptoms that resemble autism. Barile et al. (12) investigated the association between the receipt of TCVs and immune globulins early in life and neuropsychological outcomes in children at 7–10 y of age. The data were originally created by evaluating >1,000 children and their biological mothers. They found no statistically significant associations between thimerosal exposure from vaccines early in life on six of the seven variables, but there was a small but statistically significant association between early thimerosal exposure and the presence of tics in boys.
There is emerging evidence that autism may result from a maternal immune activation (MIA) during pregnancy. Several animal studies have examined the potential for prenatal viral exposure to induce aberrant behavioral outcomes in the offspring (22), and there are clinical reports of a maternal cytokine response to viral pathogens, suggesting a possible mechanism in the precipitation of these aberrant behaviors (23, 24). Maternal exposure to influenza and other viruses during pregnancy has been implicated in autism [reviewed by Zerbo et al. (25)]. In this study, pregnant dams whose infants were assigned to the 2008 group received a single influenza vaccine, containing thimerosal, to mimic vaccine recommendations for pregnant women. No evidence of either behavioral or neuroanatomical changes were observed in infants receiving a prenatal influenza vaccine, nor did our previous study identify any effects of prenatal influenza exposure on measures of early learning and cognition (17), suggesting that exposure to a single prenatal influenza TCV does not result in MIA.
In the present study, we examined social behavior in six groups of animals. Behaviors reported here were scored in animals from ∼13 to 18 mo of age. During this time, animals spent very little time engaged in negative behaviors such as Stereotypy, Rock-huddle-self-clasp, Fear-disturbed, and Withdrawal. In fact, there were virtually no instances of any stereotypy, a behavior characteristic of children with autism. Similar data were obtained in this same cohort of animals when examining behavior from ∼30 d to 12 mo of age (17). Overall, animals developed the normal repertoire of behaviors that is typical of animals of this age (26). Several primate studies have examined the effects of neurotoxicants on social behavior. Oral MeHg given prenatally alters the expression of social behavior in primates such that exposed offspring spend more time being passive and less time engaged in play behaviors with peers (26). Similarly, studies of postnatal lead exposure (27, 28) or prenatal TCDD exposure (29) have also produced a negative impact on social behavior in macaques. In contrast, exposure to low-dose TCVs via vaccination in our study did not significantly impact behavior.
There are several limitations to the present study. The 1990s Primate group was given an accelerated schedule of vaccinations due to the faster development of the visual system, pattern recognition, and object permanence in infant macaques (30, 31). It was therefore necessary to determine the appropriate timing for administering vaccines. In primates, there is a theoretical developmental ratio of 4:1, such that 4 wk of human development is comparable to 1 wk for a primate (32). Low-dose thimerosal exposure studies in primates have therefore used an accelerated schedule of exposure based on this developmental ratio (33, 34). It is possible that receiving multiple TCVs in an accelerated time frame could induce neurotoxicity in infant macaques, but this was not evident in tests of early learning and cognition (17). Likewise, in the present study, we did not find neuropathological or behavioral abnormalities in animals receiving TCVs. Neurobehavioral assessments followed very detailed protocols that have been used at the primate facility for more than three decades (35, 36). There were three testers involved in the scoring of social behavior data and each passed periodic reliability training to high standards. Therefore, although it is possible that primate behavioral scoring drifted over the course of this study (5 y), this should not have affected the group comparisons. Stereological analyses can result in biased data if suitable controls are not included. In the present study all cell number and cell size measurements were made with the person doing the measurements blind as to the experimental condition of the animal. In addition, at least two different people made the measurements to be certain of the validity of the data. Sometimes the neuroanatomical boundaries of nuclei are difficult to reliably define in all brain sections. To be certain of the neuroanatomical boundaries of the hippocampus CA1, the amygdala, and the lateral nucleus of the amygdala, we relied on the macaque primate brain atlas of Paxinos et al. (37), which allowed for a clear demarcation of the three brain regions. The present study focused on three brain regions found to be abnormal in postmortem ASD brains (cerebellum, amygdala, and hippocampus); however, the cerebral cortex has also been implicated in ASD neuropathology (38, 39). The cerebral cortex was not analyzed because there were no behavioral abnormalities observed in the present study nor was there neuropathology in the three regions examined.
In summary, analyses of postmortem brains from ASD subjects have often found decreases in Purkinje cell number (19) and cell size. We found no changes in Purkinje cell density or cell size in treated primates, and there was no difference in cerebellar calbindin, GAD-67, GFAP, or CD11b protein levels in the 1990s Primate or 2008 groups. Amygdala deficits have also been previously reported in autism. For instance, Schumann and Amaral (20) reported a 14% decrease in amygdala lateral nucleus cell number in postmortem ASD brains. We did not observe any changes in amygdala volume, lateral nucleus cell number, or cell volume in the 1990s Primate group. Postmortem studies (18) report smaller CA1 neurons in ASD cases, but in the present study we did not identify changes in CA1 cell size following administration of TCVs. Behaviors scored from ∼13 to 18 mo of age revealed that animals spent very little time engaged in autism-related behaviors. For instance, there were virtually no instances of stereotypy, a behavior characteristic of children with autism and that can be generated by administration of various CNS toxicants during this developmental period. Overall, animals in each group developed the normal repertoire of behaviors that is typical of animals of this age. Our data strongly support the conclusion that childhood TCVs do not produce ASD-like neuropathology or behavioral changes in the nonhuman primate.

Methods

Study Design.

Animal procedures followed the guidelines of the Animal Welfare Act and the Guide for Care and Use of Laboratory Animals of the National Research Council (40). All experimental protocols were approved by the University of Washington Institutional Animal Care and Use Committee. A total of 79 male infant macaques were studied in six groups: (i) Control (n = 16), animals received saline injections in place of vaccines; (ii) 1990s Pediatric (n = 12), animals received vaccines following the pediatric schedule recommended in the 1990s; (iii) 1990s Primate (n = 12), animals received vaccines recommended in the 1990s but on an accelerated schedule; (iv) TCV (n = 12), animals received all TCVs but no MMR vaccines following the accelerated schedule; (v) MMR (n = 15), animals only received the MMR vaccine but no TCVs following the accelerated schedule; and (vi) 2008 (n = 12), animals received vaccines recommended in 2008 but on an accelerated schedule. Infants were assigned to a peer group of four animals, with multiple study groups being tested each year when possible (17).

Animal Husbandry and Rearing Protocols.

All infants were nursery-raised following standardized protocols (41, 42). Details are provided in SI Methods.

Vaccine Dosing and Administration.

The vaccines used in this study are shown in Table S4. To recreate the required TCVs, thimerosal was added to the vaccines as described previously (17). Details are provided in SI Methods and Tables S5 and S6.
Table S4.
Vaccine source, EtHg content, and route of administration
VaccineTrade name (manufacturer) NDC #AbbreviationEtHg content (μg/0.5 mL dose)Administration
Hepatitis BRecombivax HB (Merck) 0006–4981-00Hep B1.98i.m.
Diphtheria, tetanus, acellular pertussisInfanrix (GlaxoSmithKline) 58160–810-46DTaP3.96i.m.
Haemophilus influenza bActHIB (Sanofi Pasteur) 49281–545-05Hib3.96i.m.
Measles mumps rubellaMMR-II (Merck) 0006–4682-00MMRNAs.c.
Inactivated polio vaccineIPOL (Sanofi Pasteur) 49281–860-10IPVNAi.m. or s.c.
RotavirusRotateq (Merck) 0006–4047-41RotaNAOral gavage
Pneumococcal 7-valent conjugate vaccinePrevnar (Wyeth) 0005–1970-67PCVNAi.m.
Hepatitis AVAQTA (Merck) 0006–4831-41Hep ANAi.m.
VaricellarVarivax (Merck) 0006–4827-00VariNAs.c.
Meningococcal polysaccharide vaccineMenomune (Sanofi Pasteur) 49281–489-05MCV3.96s.c.
InfluenzaFluzone (Sanofi Pasteur) 49281–009-50Inf3.96i.m.
Influenza (for pregnant dams only)Fluzone (Sanofi Pasteur) 49281–382-15Inf25i.m.
Modified with permission from ref. 17. NA, not applicable.
Table S5.
Primate equivalents of EtHg dosing and timing of the US pediatric vaccine recommendations in the 1990s
Vaccine dosingBirth2461548
Human (age in mo)      
 EtHg (μg) in vaccines      
  Hepatitis B × 3 doses12.512.512.5
  DTaP × 5 doses2525252525
  Hib × 4 doses25252525
  MMR × 2 doses00
 Total EtHg (μg) for infant boys12.562.562.5505025
 10th percentile weights for infant boys (kg)*2.84.45.86.8914
 μg EtHg/kg bodyweight for infant boys4.4614.2010.787.355.561.79
Primate (age in wk)      
 95th percentile weights for infant primates (kg)0.620.730.840.941.202.47
 Weight ratio infant boys:primates4.526.036.907.237.505.67
 EtHg (μg) in vaccines      
  Hepatitis B × 3 doses1.981.981.98
  DTaP × 5 doses3.963.963.963.963.96
  Hib × 4 doses3.963.963.963.96
  MMR × 2 doses00
 Total EtHg (μg) for primates vaccines1.989.99.97.927.923.96
 μg EtHg/kg bodyweight for primates3.2013.5911.818.446.611.61
Modified with permission from ref. 17.
*
Based on 10th percentile weights for infant boys from the weight-for-age percentiles from the National Center for Health Statistics, 4/20/01.
Based on 95th percentile weights for infant male macaques.
EtHg content of primate vaccines was determined by first averaging the weight ratios for human infant boys:male infant primates across the six time points of vaccine administration. The average weight ratio was 6.3:1. The EtHg content in each pediatric vaccine was then divided by 6.3 to determine the dosing of EtHg for each primate vaccine. This method provided a similar dosing of μg EtHg/kg body weight for infant boys and primates.
Table S6.
The 2008 pediatric vaccination schedule adjusted for infant primates
Prenatal*Birth2 wk4 wk6 wk12 wk15 wk18 wk26 wk52 wk
 Hep BHep B Hep B     
  RotaRotaRota     
  DTaPDTaPDTaP DTaP  DTaP
  HibHibHibHib    
  PCVPCVPCVPCV    
  IPVIPVIPV    IPV
Inf   Inf     
      MMR  MMR
      Varicella  Varicella
     Hep A Hep A  
        MCV 
The timing of all vaccine administration for primates was accelerated ∼4:1 to account for the faster developmental trajectory of infant Old World primates. DTaP, diphtheria, tetanus, acellular pertussis vaccine; Hep A, hepatitis A vaccine; Hep B, hepatitis B vaccine; Hib, Haemophilus influenza B vaccine; Inf, influenza vaccine; IPV, inactivated polio vaccine; MCV, meningococcal, vaccine; MMR, measles mumps rubella vaccine; PCV, pneumococcus vaccine; Varicella, chicken pox vaccine.
*
Pregnant dams giving birth to infants assigned to this study group received a single prenatal influenza vaccine containing 25 μg EtHg at ∼4 wk before delivery. All other dams received a single saline injection.
Influenza was administered to infants at 6 wk of age and then every 12 wk thereafter, mimicking the pediatric schedule of annual influenza vaccination. All vaccines administered were thimerosal free, except for the influenza and meningococcal vaccines, which were both formulated to contain 3.96 μg EtHg.
Meningococcal vaccine is recommended for certain high-risk groups and was therefore included to maximize EtHg exposure in animals in this group.

Assessments of Behavior.

Social behavior was evaluated daily within the home cage for each peer group from ∼12 to 18 mo of age. Each home cage contained wire mesh shelves, climbing platforms, and toys. Scoring was conducted by a blinded social tester in 5-min focal periods using a coding system of mutually exclusive and exhaustive behaviors (26). All testers were trained for 3–4 mo using the following protocol. Trainee testers score behaviors for the 5-min focal sessions along with a trained tester. This training is done for 10–15 sessions each with young infants, older infants, and young juveniles. Testers must attain a κ reliability score of 0.60 or better with their trainer with each age group being tested. All testers are retested with this procedure for reliability at 6-mo intervals. In the event of any code disagreements at or below chance levels (which was a rare occurrence), testers are retrained on the meaning of that code or codes. The three testers on this project had been social testing for 5–15 y, achieving the κ retest reliability standard at a typical value of 0.80 or better. Additional details are provided in SI Methods.

Preparation of Brain Tissues.

Brains underwent a hemidissection with alternate hemispheres processed for immersion fixation in paraformaldehyde/PBS (pH 7.4). Tissues were postfixed in the same fixative for several weeks. After cryoprotection in 20% (wt/vol) sucrose/formalin/PBS (pH 7.4) for 2–3 d at 4 °C, the forebrain was blocked in the coronal plane, frozen, and cut at 60-μm thickness on a sliding microtome.
See SI Methods for details on the preparation of brain tissues, immunohistochemistry, and immunoblot analysis.

Stereological Analysis.

All measurements were made using a Leica DMRE microscope attached to a Q-Imaging camera with Stereo-investigator software version 9.1, which was connected to a Dell Precision 450 workstation using Stereo Investigator software (MicroBrightField).

Cell counting.

For counting Nissl-positive Purkinje neurons in the cerebellum, 10 sections were examined that were spaced 1.2 mm apart from medial to lateral through an entire cerebellar hemisphere. For counting neurons in the lateral nucleus of the amygdala, seven sections were examined that were spaced 0.6 mm apart across the entire rostral-caudal dimension.

Soma size.

For measuring cell size (area), two sagittal sections (sections 100 and 300) were examined in the cerebellum, and three rostral-caudal sections were examined in the CA1 region of the hippocampus (sections 100, 200, and 300).

Volume.

For volume measurements of the cerebellum, amygdala, lateral nucleus of the amygdala, and dentate gyrus of the hippocampus, 10, 7, 9, and 8 sections, respectively, that were spaced 0.6 mm apart were measured (i.e., every 10th section). Further details are provided in SI Methods and Tables S5 and S6.

Statistical Analysis.

ANOVA was used to compare cell sizes and areas, and nuclear volumes, and multilevel modeling for analysis of behaviors among the animal groups. P < 0.05 was considered statistically significant.

SI Methods

Animal Husbandry and Rearing Protocols.

Following delivery, infants were housed in isolettes and bottle-fed by hand in the nursery until achieving temperature regulation, typically 7–10 d from birth. Infants received standard infant formula (Enfamil Premium with iron; Enfamil). Post-isolette nursery caging contained a cloth surrogate device and formula feeder until 21 d, at which time infants were rehoused in individual cages. Animal biscuits (Purina Mills) were introduced during the first month but rarely ingested until about 3 mo of age. Additional fruit treats were provided as appropriate. Animals underwent assessments for the development of neonatal reflexes, object concept permanence (OCP), discrimination learning, and behavior during the presocial living period, which have been reported separately (17). These assessments were based on the protocols developed at the Infant Primate Research Laboratory at the Washington National Primate Research Center and have been extensively published (41, 4346).
Infants underwent testing as follows: from birth to 20 d, infants were assessed for the development of neonatal reflexes and perceptual and motor skills; from postnatal day 14 to ∼3.5 mo of age, infants were examined for the development of OCP; from ∼3 to 6 mo of age, animals underwent discrimination learning assessments; from ∼5 to 8 mo of age, animals were assessed for learning set development; and from 30 d to 12 mo of age, animals underwent assessments of behavior before group living. These developmentally appropriate tests are measures of neurodevelopment, learning, cognitive abilities, and social behavior in young macaques (45). At ∼13 mo of age, animals were transferred to juvenile caging where they were group housed (n = 4 males per group) with animals from within their peer group for the duration of the study. All subsequent behavioral data were collected while animals were in their home cage.

Vaccine Dosing and Administration.

The concentration of EtHg in vaccine aliquots was periodically verified throughout the study using an independent testing laboratory (Quicksilver Scientific). All infants received either a vaccine or saline injection, administered i.m. or s.c. in a 0.5-mL volume, or orally, depending on recommended procedures, according to study group assignment. The vaccine dosing schedule was adjusted for all but one group (1990s Pediatric) to accommodate the approximate 4:1 developmental trajectory of infant Old World monkeys (4750). Thus, when the human schedule called for vaccines to be administered at birth, 2 mo, 4 mo, 6 mo, 15 mo, and 48 mo, the timing of the primate vaccine schedule was accelerated fourfold and given at birth, 2 wk, 4 wk, 6 wk, 15 wk, and 52 wk. Vaccines were administered according to the schedule used in the 1990s (Table S5) or the expanded vaccine schedule from 2008 (Table S6), when fewer vaccines contained thimerosal. To follow the recommendation that pregnant women receive an influenza vaccine during gestation, pregnant dams of infants assigned to the 2008 group also received a single influenza vaccine containing 25 μg EtHg at ∼4 wk before delivery. All other dams received a saline injection.

Assessments of Behavior.

Order of testing was randomized for each session. Scored behaviors included the following: Passive, Explore, Play, Sex, Aggression, Withdrawal, Fear-disturbed, Rock-huddle-self-clasp, and Stereotypy, and could be scored as either a social interaction or a nonsocial behavior (Table S1). Although negative behaviors (Withdrawal, Fear-disturbed, Rock-huddle-self-clasp, and Stereotypy) are considered part of the typical repertoire of behaviors for macaques of this age, significant increases in these behaviors could be considered “ASD-like behaviors” as the definition of ASD includes stereotyped repetitive patterns of behavior (2). Additional behaviors scored during each 5-min focal period, but not included in the analyses, included eating, drinking, grooming, and scratching. A mean duration and frequency was computed for each 30-d period for each of these nine behaviors. The first 30-d average for each animal began at initiation of social living (∼12 mo of age), and the final 30-d average was the last full 30-d period before necropsy (i.e., the final period was excluded if it contained fewer than 30 d). Animals averaged 5.78 ± 1.11 (mean ± SD) 30-d periods in social living.
Before fitting models to the behavioral data, descriptive statistics for behavior duration and frequency were examined. Means and SDs for durations and frequencies are displayed in Table 2. Since duration and frequency closely mirrored each other, only duration was used as an outcome in the analytic models. Also, because the duration of the negative behaviors was low overall, Stereotypy, Rock-huddle-self-clasp, Fear-disturbed, and Withdrawal were summed for each animal, as were durations of the positive behaviors (Aggression, Sex, and Play). Thus, for both nonsocial behavior (involving no other animal) and social behavior (involving one or more animals), there were four outcomes used in the analysis: passive, explore, positive, and negative. Duration values were natural log-transformed to reduce the possibility of disproportionate influence from extreme values.
Linear mixed models were fit to describe behaviors following recommendations for longitudinal model building (41). Linear mixed models accommodate data with multiple observations from the same subject and unequal numbers of observations per subject, both of which were characteristics of the present data. In addition, time can be treated as a continuous variable allowing it to be flexibly modeled to capture the trajectory of change in the outcome across the course of the study and interactions between experimental conditions and the trajectory of change. Before adding animal-level variables, a series of unconditional growth models were fit, which included an unconditional mean (i.e., no change), linear, and quadratic models. The best growth model for a putative outcome was determined by comparing unconditional growth models using the Akaike information criterion (51). Time was centered at the first month of social living. Among the social data, the best growth model was the unconditional means model for passive, explore, and negative behaviors, indicating that these behaviors remained constant across social living. The best growth model for positive behaviors was the quadratic model. Among nonsocial data, the best growth model for passive and explore was the linear model. The best growth model for negative behaviors was the unconditional means and the best growth model for positive behaviors was the quadratic model. After establishing the growth model for each outcome, the intervention condition main effect was added to the model and all interactions between the intervention condition, and time parameters were added to the models. In addition, age at the beginning of social living (mean = 365.66 d, SD = 35.33) was included in each model. A false discovery rate correction was applied to each parameter across the eight models. In the event of either a significant group or a group × time interaction effect, simple slope comparisons between the control group and each of the other groups were used to assess whether there were differences at initiation of social living and at the sixth month (the median number of months in social living) (52). These models were previously used for the analysis of macaque behavior (17).

Preparation of Brain Tissues.

The entire cerebellar hemisphere was cut in the sagittal plane. Every section was saved and if not used for Nissl/immunohistochemical staining, it was put into a labeled (brain # and section #) polypropylene tube and archived for future use. Some brains were not available for stereological analysis due to unsatisfactory cryoprotection.

Immunohistochemistry.

Free-floating sections were blocked with 4% (wt/vol) goat serum/PBS plus 0.2% Triton X-100 and incubated overnight with antibodies against calbindin (1:20,000 dilution; Swant Laboratories), GFAP (1:4,000 dilution; Abcam), CD11b (1:4,000 dilution; Abcam), and doublecortin (1:500 dilution; Abcam). ABC reagents (Vector Laboratories) and SigmaFast DAB peroxidase substrate (Sigma-Aldrich) were also used. Subsequently appropriate secondary antibodies were used that are complementary to the primary antibodies. Sections were counterstained for Nissl (0.09% Cresyl violet or neutral red).

Immunoblot analysis.

Frozen cerebellar tissue (∼100 mg) was dissected from the posterior lobe. The tissue was homogenized in lysis buffer, 1% SDS, 1× PBS, and Complete Protease Inhibitor Mixture (Sigma), using a Diax 900 homogenizer (Sigma). After homogenization, the homogenate was centrifuged at 8,100 × g for 10 min, and the resulting fractions were collected. Protein levels were quantified using the BCA Kit (Pierce) with BSA standards and analyzed by immunoblot. The supernatant was used to separate proteins by SDS/PAGE [Tris-glycine 4–20% (wt/vol) gradient precast gels; Bio-Rad] and subjected to immunoblot analysis with the following antibodies: Iba1 (1:2,000 dilution; Abcam); GFAP (1:5,000; Abcam); calbindin (1:2,000 dilution; Waco Laboratories); GAD-67 (1:750 dilution; Abcam); and β-actin (1:10,000 dilution; Cell Signaling). Subsequently appropriate secondary HRP-conjugated antibodies (Vector Laboratories) were used that are complementary to the primary antibodies. Immunoblot signals were visualized with chemiluminescence. For densitometry analysis of the protein bands for different antibodies, the data were analyzed by using optical density measurement tools in ImageJ software (National Institutes of Health).

Stereological analysis.

Three brain regions were analyzed.
i)
Cerebellum: The optical fractionator method was used to estimate the number of Purkinje neurons in the cerebellum at a magnification of 20× [as described by Pakkenberg et al. (53)]. More than 400 cells were counted per brain. The optical fractionator counting frame was 425 × 275 μm, and the sampling grid was 3,000 × 3,000 μm. A Purkinje cell was defined as a large-sized soma in the Purkinje cell layer that occurred within the z-plane of the counting frame (10 μm, with 1-μm upper and lower guard zones).
ii)
Hippocampus: Doublecortin immunostaining was used to identify newborn dentate granule cell neurons. The counting frame was 100 × 100 μm and the sampling grid was 400 × 400 μm, and cells were identified using 40× magnification. Eight sections were examined at an interval of every 10th section. More than 500 cells were counted per brain.
iii)
Amygdala lateral nucleus: Neuron number was estimated using 40× magnification. The counting frame was 200 × 125 μm and the sampling grid was 800 × 800 μm. Every 10th section was examined with seven sections per brain. More than 400 cells were counted per brain, and neurons with a nucleolus in focus within the counting frame were counted.

Estimation of cell area.

The cross-sectional areas of neurons were measured in the cerebellum, hippocampus, and lateral nucleus of the amygdala using the Nucleator method (54). Each time a neuron was encountered with a clearly identified nucleolus in focus within the z-plane, the nucleolus was marked, and a grid of two radially extended lines emanated from the nucleolus to the cell membrane. The four points at which each line intersected the neuronal cell membrane were marked, to calculate the neuronal cross-sectional area.
i)
Cerebellum: To estimate the cell area of Purkinje neurons, a magnification of 40× was used, and cell area was determined in both calbindin-positive and calbindin-negative (i.e., Nissl-positive) Purkinje cells. The counting frame was 200 × 125 μm and the sampling grid was 1,250 × 1,250 μm; two sections per brain were examined at medial and lateral portions of the cerebellum. From 100 to 150 calbindin-positive and Nissl-positive cells were measured per brain.
ii)
Hippocampus: Two configurations were used to measure the cell area of the CA1 neurons. Leica DMRE Microscope configuration: Brain sections were analyzed under the microscope using 40× magnification with a counting frame of 100 × 100 μm and a sampling grid of 250 × 250 μm. Two sections were examined per brain, and 100 to 200 cells were measured per brain. Hamamatsu NanoZoomer 2.0HT configuration: Brain sections were scanned in eleven 1-μm layers (z-planes). A digital 63× lens was used for cell soma measurements. Three sections and ∼250 to 350 cells were measured per brain. Two independent researchers measured a mean neuronal cross-sectional area for each brain and were blind to animal groups. The percent difference between the independently measured means did not exceed 5.5% wt/vol.
iii)
Lateral nucleus: To estimate the cell area of neurons in the lateral nucleus of the amygdala, 40× magnification was used with a counting frame of 200 × 125 μm and a sampling grid of 450 × 600 μm. More than 200 cells were measured per brain in one section located at the rostral-caudal center of the nucleus.

Estimation of volume.

The volume of the cerebellum, amygdala, lateral nucleus of the amygdala, and dentate granule cell layer of the hippocampus was measured using the Cavalieri method.
i)
Dentate gyrus: To estimate the volume of dentate gyrus of the hippocampus, outlines were drawn around the structure at 1.25× magnification. Eight 60-μm-thick sections were examined in each brain, spaced 10 sections apart.
ii)
Amygdala: The outline of the amygdala was drawn at 1.25× magnification using the brain atlas of Paxinos et al. (37). The outlines in nine coronal sections were spaced 10 sections apart through the rostral-caudal extent of the nucleus.
iii)
Amygdala lateral nucleus: The outline of the lateral nucleus of the amygdala was drawn at 1.25× magnification according to the brain atlas of Paxinos et al. (37) in seven coronal sections through the rostral-caudal extent of the nucleus.

Acknowledgments

We thank the staff at the Infant Primate Research Laboratory at the Washington National Primate Research Center, including Dr. Robert Murnane, Dr. Keith Vogel, Cliff Astley, Dr. Tom Burbacher, Debra Glanister, Elaine Adkins, Megan Rulian, Kelly Morrisroe, Caroline Kenney, Noelle Liberato, India Tindale, Kristen Watkins, Brenda Crouthamel, and Mac Durning. We thank Dr. Tricia Coakley at the University of Kentucky for preparation of thimerosal-containing vaccines (TCVs), and the California National Primate Research Center for providing pregnant dams for this study. We thank the following for their generous financial support: The Ted Lindsay Foundation, SafeMinds, National Autism Association, and the Johnson and Vernick families. This work was also supported by WaNPRC Core Grant RR00166 and CHDD Core Grant HD02274.

Supporting Information

Supporting Information (PDF)
Supporting Information

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 112 | No. 40
October 6, 2015
PubMed: 26417083

Classifications

Submission history

Published online: September 28, 2015
Published in issue: October 6, 2015

Keywords

  1. pediatric vaccines
  2. autism
  3. rhesus macaque
  4. thimerosal
  5. neuropathology

Acknowledgments

We thank the staff at the Infant Primate Research Laboratory at the Washington National Primate Research Center, including Dr. Robert Murnane, Dr. Keith Vogel, Cliff Astley, Dr. Tom Burbacher, Debra Glanister, Elaine Adkins, Megan Rulian, Kelly Morrisroe, Caroline Kenney, Noelle Liberato, India Tindale, Kristen Watkins, Brenda Crouthamel, and Mac Durning. We thank Dr. Tricia Coakley at the University of Kentucky for preparation of thimerosal-containing vaccines (TCVs), and the California National Primate Research Center for providing pregnant dams for this study. We thank the following for their generous financial support: The Ted Lindsay Foundation, SafeMinds, National Autism Association, and the Johnson and Vernick families. This work was also supported by WaNPRC Core Grant RR00166 and CHDD Core Grant HD02274.

Notes

This article is a PNAS Direct Submission. M.S. is a guest editor invited by the Editorial Board.
See Commentary on page 12236.

Authors

Affiliations

Bharathi S. Gadad
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Wenhao Li
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Umar Yazdani
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Stephen Grady
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Trevor Johnson
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Jacob Hammond
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Howard Gunn
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Britni Curtis
Infant Primate Research Laboratory, Washington National Primate Research Center, Seattle, WA 98195;
Chris English
Infant Primate Research Laboratory, Washington National Primate Research Center, Seattle, WA 98195;
Vernon Yutuc
Infant Primate Research Laboratory, Washington National Primate Research Center, Seattle, WA 98195;
Clayton Ferrier
Infant Primate Research Laboratory, Washington National Primate Research Center, Seattle, WA 98195;
Gene P. Sackett
Infant Primate Research Laboratory, Washington National Primate Research Center, Seattle, WA 98195;
Department of Psychology, University of Washington, Seattle, WA 98195;
C. Nathan Marti
Independent Consultant, Austin, TX 78711;
Present address: The School of Social Work, University of Texas at Austin, Austin, TX 78712.
Keith Young
Department of Psychiatry and Behavioral Science, Texas A&M Health Science Center & Central Texas Veterans Health Care System, Temple, TX 76504;
Laura Hewitson
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Johnson Center for Child Health & Development, Austin, TX 78701
Dwight C. German2 [email protected]
Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Notes

2
To whom correspondence should be addressed. Email: [email protected].
Author contributions: G.P.S., K.Y., L.H., and D.C.G. designed research; B.S.G., W.L., U.Y., S.G., T.J., J.H., H.G., B.C., C.E., V.Y., C.F., and D.C.G. performed research; B.S.G., U.Y., G.P.S., C.N.M., and D.C.G. analyzed data; and B.S.G., G.P.S., C.N.M., L.H., and D.C.G. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    Administration of thimerosal-containing vaccines to infant rhesus macaques does not result in autism-like behavior or neuropathology
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 40
    • pp. 12223-E5555

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